Synthesis, Characterization, and Biological Properties of Steroidal Ruthenium(II) and Iridium(III) Complexes Based on the Androst-16-en-3-ol Framework

Article abstract of DOI:10.1021/acs.inorgchem.9b02402

A range of novel cyclometalated ruthenium(II) and iridium(III) complexes with a steroidal backbone based on androsterone were synthesized and characterized by NMR spectroscopy and X-ray crystallography. Their cytotoxic properties in RT112 and RT112 cP (cisplatin-resistant) cell lines as well as in MCF7 and somatic fibroblasts were compared with those of the corresponding nonsteroidal complexes and the noncyclometalated pyridyl complexes as well as with cisplatin as reference. All steroidal complexes were more active in RT112 cP cells than cisplatin, whereby the cyclometalated pyridinylphenyl complexes based on 5c showed high cytotoxicity while maintaining low resistant factors of 0.33 and 0.50.

Full text of DOI:10.1021/acs.inorgchem.9b02402

Article
pubs.acs.org/IC
Cite This: Inorg. Chem. XXXX, XXX, XXX−XXX
Synthesis, Characterization, and Biological Properties of Steroidal
Ruthenium(II) and Iridium(III) Complexes Based on the Androst-16-
en-3-ol Framework
Vanessa Koch,† Anna Meschkov,†,‡ Wolfram Feuerstein,§ Juliana Pfeifer,‡ Olaf Fuhr,∥ Martin Nieger,⊥
,†,#
Ute Schepers,†,‡ and Stefan Brase*
̈
†Institute of Organic Chemistry (IOC), Karlsruhe Institute of Technology (KIT), Fritz-Haber-Weg 6, 76131 Karlsruhe, Germany
‡Institute of Functional Interfaces (IFG), Karlsruhe Institute of Technology (KIT), Hermann von Helmholtz Platz 1, 76344
Eggenstein-Leopoldshafen, Germany
§Institute of Inorganic Chemistry, Division Molecular Chemistry, Karlsruhe Institute of Technology (KIT), Engesserstr. 15, 76131
Karlsruhe, Germany
∥Institute for Nanotechnology (INT) and Karlsruhe Nano Micro Facility (KNMF), Karlsruhe Institute of Technology (KIT),
Hermann von Helmholtz Platz 1, 76344 Eggenstein-Leopoldshafen, Germany
⊥Department of Chemistry, University of Helsinki, P.O. Box 55, 00014 Helsinki, Finland
#Institute of Toxicology and Genetics, Karlsruhe Institute of Technology (KIT), Hermann-von-Helmholtz-Platz 1, D-76344
Eggenstein-Leopoldshafen, Germany
S
* Supporting Information
ABSTRACT: A range of novel cyclometalated ruthenium(II)
and iridium(III) complexes with a steroidal backbone based
on androsterone were synthesized and characterized by NMR
spectroscopy and X-ray crystallography. Their cytotoxic
properties in RT112 and RT112 cP (cisplatin-resistant) cell
lines as well as in MCF7 and somatic fibroblasts were
compared with those of the corresponding nonsteroidal
complexes and the noncyclometalated pyridyl complexes as
well as with cisplatin as reference. All steroidal complexes
were more active in RT112 cP cells than cisplatin, whereby
the cyclometalated pyridinylphenyl complexes based on 5c
showed high cytotoxicity while maintaining low resistant
factors of 0.33 and 0.50.
ruthenium complexes of the type [Ru(η6-arene)Cl2(PTA)]
(PTA = 1,3,5-triaza-7-phosphatricyclo[3.3.1.1]decane) also
INTRODUCTION
■
Over the past 60 years, the interest in steroid-bearing
show promising anticancer properties and are therefore
transition-metal complexes has increased continuously.1 In
broadly investigated.15−17 By connecting the metal center to
the late 1970s, the biological application of these complexes
was discovered, enabling new perspectives for metal-containing
a steroidal backbone as shown in complexes 1 and 2
approaches, e.g., in the treatment of cancer.1 Although
(Figure 1), biological properties can be tuned. Since the
platinum-based anticancer complexes2 such as cisplatin3,4 or
steroidal framework enables the binding to steroid receptors,
oxaliplatin5,6 are worldwide recognized for the treatment of
cell penetration can be improved. It has been also
demonstrated that by the incorporation of a C-3 modified
cancer, still some drawbacks remain. Diverse side effects as well
cholesterol ruthenium(III) complex 2 into a liposome bilayer,
as multifactorial drug resistance mechanisms, including, e.g.,
enhanced DNA repair, increased drug efflux, and detoxifica-
the ruthenium moiety was protected from degradation and the
tion, cause severe limitations in therapeutic use.7,8 In order to
cellular uptake was favored. When integrated into a biomimetic
membrane, the complex was found to be 6-fold times more
circumvent intrinsic and acquired resistance and to reduce side
active against the MCF-7 cell line (breast cancer) than the
effects, other transition-metal based anticancer agents have
corresponding nonsteroidal complex.18 Moreover, Jaouen et
been explored.9 On the basis of ruthenium(III), KP101910 and
NAMI-A11 (Figure 1) were developed, whereby both drugs
already passed phase I of the clinical trials.12−14 Ruthenium-
(III) probably serves as a pro-drug and is reduced within the
cell to the active ruthenium(II) complex. Noteworthy,
al.19,20 and later Hannon et al.21−24 could show that a sufficient
Received: August 9, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.9b02402
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Figure 1. Currently investigated Ru(III)- and Ru(II)-anticancer drugs and ruthenium complexes based on an androgen (1) and a cholesterol (2)
framework.
Scheme 1. Synthesis of the Steroidal Pyridine-Containing Ligands 5 Starting from epi-Androsterone (3) via a Two-Step
Procedure31
recognition of steroidal receptors is retained if the organo-
metallic site is attached at the end of a rigid spacer such as an
ethynyl group at C-17 of an estradiol or testosterone derivative.
In this context, Ruiz et al. found that the androgen-containing
ruthenium(II) complex 1 was 8-fold more active than cisplatin
in T47D cell lines (breast cancer).25,26 Lv et al. also reported
an enhancement of the antiproliferative activity of an N-
heterocyclic carbene ruthenium complex in MCF-7 cells by
conjugating a 17α-ethynyl testosterone via a disulfide linkage.27
Compared to the number of studies on the anticancer
activity of ruthenium complexes, only a few reports regarding
the anticancer activity of iridium(III) complexes have been
published.25,28−30 Nevertheless, Sadler et al. showed that the
biological activity of pentamethylcyclopentadienyl (Cp*)Ir-
(III) complexes was increased by the incorporation of phenyl
substituents. This resulted in an enhanced cellular accumu-
lation due to the higher hydrophobicity of these complexes.
Furthermore, the substitution of N,N-ligands by C,N-chelating
ligands was shown to improve antiproliferative activity.
Motivated by these results, we envisioned to investigate the
chemical, spectroscopic, and biological properties of novel
ruthenium(II) and iridium(III) complexes based on epi-
androsterone with the metal center located closer to the
steroidal backbone compared to previous examples.25,26
B
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Scheme 2. Synthesis of Novel Cycloruthenated Ruthenium(II) Complexes 6, 7, and 8
Figure 2. Molecular structure of the pyridylphenyl ruthenium(II) complex 7 (displacement parameters are drawn at 50% probability level).
Characteristic bond lengths: Ru−N 2.097(3) Å; Ru−C 2.047(3) Å; Ru−Cl 2.4253(7) Å; Ru−Ccymene 2.154(3)−2.290(3) Å; Ru−Ccymene/centroid
1.706(3) Å. Selected bond angles: N−Ru−C 77.72(12)°, N−Ru−Cl 86.98(3)°, Cl−Ru−C 85.76(9)°, N−Ru−cymenecentroid 132.3(1)°, Cl−Ru−
cymenecentroid 126.6(1)°, C−Ru−cymenecentroid 130.3(1)°.
tion afforded the 2′-pyridinyl derivative 5a, 3′-pyridinyl
derivative 5b, or 4-(pyridin-2′-yl)phenyl derivative 5c in
good yields ranging from 60−74%. By washing the obtained
products 5 with n-hexane, traces of remaining stannanes could
be removed, which was crucial with regard to biological tests.
Since numerous procedures exist in the literature for the
synthesis of cyclic ruthenium(II) complexes of 2-phenyl-
pyridines,32−34 we tried analogous reaction conditions with 2-
(4-bromophenyl)pyridine. With one equivalent of dimeric
ruthenium precursor [Ru(η6-para-cymene)Cl2]2 and two
equivalents of the ligand in the presence of four equivalents
of KOAc in MeOH, the Ru(II) complex was formed after
stirring at room temperature for 24 h. After flash column
chromatography on silica gel, the ruthenium(II) complex 6 was
isolated with 67% yield (Scheme 2). Applying the same
reaction conditions, the synthesis of the phenylpyridinyl
ruthenium(II) complex 7 and the pyridinyl ruthenium(II)
complex 8 succeeded in moderate yields starting from their
ligands 5c or 5a (Scheme 2).
DESIGN AND SYNTHESIS OF THE NEW
RUTHENIUM COMPLEXES
■
In order to bring the steroidal backbone in close proximity to
the metal center, we aimed to modify C-17 of epi-androsterone
(3) in such a manner that the complexation of ruthenium(II)
and the iridium(III) is feasible either by an N-pyridine moiety
or by κ2-N,C-cyclometalation. Therefore, different pyridine
substituted androsterone derivatives (5a, 5b) and a 4′-(2-
pyridinyl)phenyl derivative (5c) were synthesized. As
previously shown by our group, pyridine containing sub-
stituents are best introduced by the Stille cross-coupling
reaction.31 Starting from epi-androsterone (3), the desired
ligands were easily accessible by a two-step procedure
(Scheme 1). Hence, epi-androsterone (3) was treated with
hydrazine to form the hydrazone giving either the alkenyl
iodide 4a by adding iodine in the presence of triethylamine or
the alkenyl bromide 4b by adding NBS with pyridine as a base.
The following palladium-catalyzed Stille cross-coupling reac-
C
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Table 1. Experimental Proton and Carbon Resonances of the Diastereomers (R-Ru)-8 and (S-Ru)-8 and Their Experimental
Differences of the Chemical Shifts Δδ = δS−δR As Well As Their Differences calΔδ= δ(S)−δ(R) Calculated on the TPSSh/def2-
a
TZVPP Level of Theory Using bp86/def2-TZVPP Structures
(R)-diastereomer
(S)-diastereomer
13C
1H
δR [ppm]
Δ
δ(S‑R)[ppm]
δS [ppm]
calΔδ[ppm]
δR [ppm]
Δδ[ppm]
δS[ppm]
calΔδ[ppm]
16-Cq−Ru
17-Cq−Pyr
13-Cq
208.7
150.4
44.8
16.6
44.8
+1.0
+0.3
−0.5
+0.8
−0.5
209.7
150.7
44.3
17.4
44.3
+0.6
+0.2
−0.2
+1.7
−0.3
18-CH3
0.84
3.11
2.50
n.d.
+0.09
−0.13
+0.34
n.d.
0.93
2.98
2.84
n.d.
+0.12
−0.25
+0.33
−0.01
15α-CH2
15β-CH2
14-CH
58.5
−0.2
58.3
+1.1
a
By comparing the calculated with experimental differences in chemical shifts, the diastereomers (R-Ru)-8 and (S-Ru)-8 were assigned. n.d. = not
determined. See text for computational details.
Figure 3. 1H NMR spectra (CDCl3, 500 MHz, r.t.) of the diastereomers (R-Ru)-8 (top) and (S-Ru)-8 (bottom). Significantly different shifts of the
two diastereomers are highlighted (blue, 15-CH2; yellow, cymene; green, 18-CH3).
1
By recording H and 13C NMR, IR, and FAB mass spectra
the complexes 6, 7, and 8 were successfully characterized. The
Ru metal takes a pseudotetrahedral “piano stool” coordination
geometry generating a new stereogenic center. Hence, most of
the NMR resonances of the pyridinylphenyl ruthenium(II)
complex 7 in d1-chloroform were duplicated (see Supporting
Information). DFT calculations on the BP8635,36/def2-
TZVPP37 level reveal the two diastereomers to differ only
5.6 kJ/mol in Gibbs free energy. Accordingly, both
diastereomers were formed in comparable amounts as
D
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evidenced by NMR signal intensities showing that no
diastereomeric induction for cycloruthenation occurred.
Furthermore, a single crystal suitable for X-ray crystallography
was obtained confirming the stated molecular structure for the
R-diastereomer as depicted in Figure 2. The coordination
geometry of the Ru(II) center shows the expected pseudote-
trahedral geometry. The N−Ru−C angle of 77.72(12)° is
significantly smaller than the N−Ru−Cl (86.98(3)°) and the
Cl−Ru−C (85.76(9)°) angles, which is in agreement with
reported nonsteroidal cycloruthenated 2-phenylpyridinyl com-
plexes.38 In comparison with nonsteroidal complexes reported
in the literature,38,39 ruthenium(II) complexes 7 show similar
bond angles and bond lengths, whereby Ru−X bond lengths
(X = N, Cl, C) are slightly longer and bond angles Y−Ru−Z
(Y≠ Z = N, Cl, C) slightly smaller. Unfortunately, we were not
able to assign the crystal structure to one of the NMR signal
sets, since the NMR chemical shifts of the diastereomers are
too similar, as predicted by NMR shielding calculations (see
Supporting Information).
shielding. It is noteworthy that for diastereomer (S-Ru)-8, the
individual 15-CH2 resonances were closer to each other than
those for the diastereomer (R-Ru)-8, which was predicted by
our chemical shielding calculations as well. This behavior is
caused by the chlorido substituent: The nonbonding electrons
lead to a shielding of spatially close protons by n−σ*
interactions.41 Hence, the β-proton of diastereomer (R-Ru)-8
is shifted to higher fields compared to its (S-Ru)-8 counterpart.
The same held true for the α-proton of (S-Ru)-8, which is,
however, less pronounced.
We were able to obtain a crystal structure of the
(R)-diastereomer of 8 (Figure 4) confirming the molecular
Fortunately, in the case of the 2-pyridinyl ruthenium(II)
complex 8, the diastereomers could be separated by column
chromatography on silica, whereby both diastereomers (R-
Ru)-8 and (S-Ru)-8 were formed in equal amounts according
1
to the integration of the crude H NMR spectrum and could
be isolated in comparable amounts. As for complex 7, by
optimizing the molecular structures of both diastereomers at
the BP8635,36/def2-TZVPP37 level of theory, we could show
that none of the two diastereomers was noticeably
thermodynamically favored standing in line with the nearly
equimolar ratio of the isolated product. Both diastereomers
showed nearly the same Gibbs free energies differing only by
3.2 kJ/mol in favor of the (S)-diastereomer. In addition, we
calculated 1H and 13C NMR shifts employing different density
functionals using the optimized structures of both diaster-
eomers to be compared with the experimental resonances. The
TPSSh functional40 turned out to yield the best accordance
with the experimental NMR shift differences between
(R-Ru)-8 and (S-Ru)-8 (see Supporting Information). This
allowed for the assignment of the obtained NMR spectra to the
two diastereomers (Table 1).
Figure 4. Molecular structure of the 2-pyridinyl ruthenium(II)
complex (R-Ru)-8 (displacement parameters are drawn at 50%
probability level). Characteristic bond lengths: Ru−N 2.078(10) Å;
Ru−C 1.931 Å; Ru−Cl 2.415(3) Å; Ru−cymene 2.130(16)−
2.341(14) Å; Ru−cymene/centroid 1.729 Å. Selected bond angles:
N−Ru−C 76.3(4)°, N−Ru−Cl 85.2(2)°, Cl−Ru−C 86.4(3)°, N−
Ru−cymenecentroid 132.33°, Cl−Ru−cymenecentroid 126.60°, C−Ru−
cymenecentroid 130.34°.
structure and the correct assignments of the diastereomers
based on the calculated chemical shifts. In contrast to the
phenylpyridinyl ruthenium(II) complex 6 and its steroidal
counterpart 7, the Ru−C bond of (R-Ru)-8 is significantly
shorter (1.931 Å vs 2.062 Å of 639/2.047 Å of 7). This is an
indication for the inferior electron donating ability of the
cyclopentenido moiety of the steroidal D-ring compared to
phenyl. To the best of our knowledge, the herein presented
ruthenium complex 8 is the first example of a cyclopentenido-
pyridinyl ruthenium(II) complex.42,43
Furthermore, comparable iridium(III) complexes were
synthesized by applying similar reaction conditions and
[IrCp*Cl2]2 as a metal precursor. The three cyclometalated
Ir(III) complexes 9, 10, and 11 (Scheme 3) were synthesized
in overall good yields, whereby the diastereomers of 10 and 11
were formed in equal amounts giving two sets of signals in the
1H and 13C NMR spectra. Unfortunately, a separation of the
diastereomers 11 via column chromatography was not
successful.
For comparison with the cycloruthenated complexes 7 and 8
as well as for the analogous iridium(III) complexes 10 and 11,
(17-(3′-pyridinyl)androsten)dichloride ruthenium(II) com-
plex 12 and the corresponding iridium(III) complex 13 were
synthesized by stirring two equivalents of the ligand 5b and
one equivalent of the dimeric metal precursor in dichloro-
methane (Scheme 4). After precipitation with n-hexane, the
metal complexes 12 and 13 were obtained in good yields.
We were able to obtain crystal structures suitable for crystal
structure analysis for both 3-pyridyl complexes 12 and 13
The corresponding 1H NMR spectra and the relevant
assignments are depicted in Figure 3. The stacked NMR
spectra show no differences in chemical shifts for the proton
resonances of the pyridinyl moiety. The aromatic and aliphatic
proton resonances of the cymene ligand on the other hand,
were clearly shifted similar to the three isopropyl resonances of
(S-Ru)-8 that appear at lower chemical shifts compared to the
ones of (R-Ru)-8. Also, the resonances of the steroidal
backbone close to the ruthenium center are affected by the
different electronic environments of the two diastereomers. For
example, in case of the diastereomer (R-Ru)-8, the methyl
group and the chlorido substituent were located on the same
side, resulting in a chemical shift of δ = 0.94 ppm, while the
resonances of the diastereomer (S-Ru)-8 are shifted upfield to
δ = 0.84 ppm. Furthermore, the two diastereomeric 15-CH2
resonances were influenced by the coordination to the
pseudotetrahedral ruthenium center, whereby both signal sets
were shifted downfield compared to those of the steroidal
ligand 5b. NOESY experiments and the evaluation of the
coupling constants allowed the assignment of the more
shielded signals to the 15β-CH2 protons, while the signals
that arise more downfield belong to the 15α-CH2 protons,
being in accordance with our calculations of the chemical
E
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Scheme 3. Synthesis of Novel Cyclometalated Iridium(III) Complexes 9, 10, and 11
Scheme 4. Synthesis of the (17-(3′-Pyridinyl)androstenido)dichloride Ruthenium(II) Complex 12 and the Corresponding
Iridium(III) Complex 13
Figure 5. Molecular structure of the 3-pyridyl ruthenium(II) complex 12 (left) and the 3-pyridyl iridium(III) 13 (right). The cymene ligand of
ruthenium(II) complex 12 is disordered. Selected bond lengths for 12/13: Ru−N 2.125(3) Å; Ru−Ccymene 2.159(10)−2.231(10) Å; Ru−Cl(1)
2.419 Å; Ru−Cl(2) 2.404 Å/Ir−Cl(1) 2.395(5) Å; Ir−Cl(2) 2.408(6) Å; Ir−N 2.090(5) Å; Ir−CCp* 2.11(2)−2.26(2) Å; Ir−CCp*/centroid 1.788 Å.
Selected bond angles for 12/13: Cl(1)−Ir−Cl(2) 89.2(2)°; N−Ir−Cl(1) 85.9(5)°; N−Ir−Cl(2) 85.6(5)°; N−Ir−CCp*/centroid 125.7(2)°; Cl(1)−Ir−
CCp*/centroid 127.4(8)°; Cl(2)−Ir−CCp*/centroid 128.9(5)°/Cl(1)−Ru−Cl(2) 87.4(7)°; N−Ru−Cl(1) 85.2(1)°; N−Ir−Cl(2) 84.9(0)°.
F
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(Figure 5), confirming their molecular structure and the
pseudotetrahedral coordination geometry around the metal.
Interestingly, in the solid state, the nitrogen atom of the pyridyl
moiety points toward the 18-methyl group and the metal
atoms were located on the upper side of the steroidal
framework. It is noteworthy that the ruthenium(II) complex
12 shows almost no distortion (6.81°) in contrast to the free
ligand 5b (see Supporting Information for the crystal
structure) and the iridium(III) complex 13 whose pyridine
units are twisted to the D-ring plane with a torsion of 32.9(3)°
or 29.1(8)°.
and (R)-diastereomers), 11, 12, and 13 was in the same order
of magnitude as that of the corresponding free steroidal ligands
(5a and 5b), with IC50 values in the range between 2.5 and 7.5
μM for both cell lines. However, the steroidal ligand 5c
demonstrated high biocompatibility (IC50 > 50 μM for both
cell lines), whereas the corresponding Ru(II) (7) and Ir(III)
(10) complexes showed promising antiproliferative effects in
both cell lines. This indicates that the cisplatin resistance was
successfully overcome with IC50 values of 1 μM for RT112 cP
cells and very low resistance factors of 0.33 and 0.5,
respectively. Although the nonsteroidal complexes 6 and 9
were also more toxic (IC50 5−8 μM) compared to the free
nonsteroidal (4‑bromophenyl)pyridine ligand (IC50 > 50 μM),
the steroidal complexes 7 and 10 were significantly more
effective with considerably lower IC50 values and resistance
factors. For the MCF-7 cells, all the steroid complexes
demonstrated a high cytotoxic activity with IC50 values in
the range of 2.5−8.5 μM, significantly more effective as
cisplatin with an IC50 value of 22.5 μM. The Ru(II) complex 7
was slightly more active in comparison to the corresponding
ligand 5c (IC50 values 2.5 and 6.6 μM, respectively). Moreover,
the cytotoxic activity of the majority of the compounds was
lower for NHDF compared to the cancer cell lines, with
especially high IC50 values for the complexes 12 and 13 (>50
μM), suggesting a therapeutic window with a selectivity for
cancer cells.
BIOLOGICAL ACTIVITY AND CYTOTOXICITY
STUDIES
To evaluate the cytotoxicity of the compounds, an in vitro
■
MTT assay was performed (Table 2). The MTT (3-(4,5-
Table 2. IC50 Values and Resistance Factors (RF,
IC50(resistant)/IC50(sensitive)) of the Ru(II) and Ir(III)
Complexes, Free Ligands, and Cisplatin (μM)
RT112 cP
entry
1
compound
RT112
>50.0
(RF)
MCF-7 NHDF
(4-bromophenyl)
pyridine
>50.0
>50.0
>50.0
2
3
4
5
Ru(II) complex 6
Ir(III) complex 9
ligand 5a
Ru(II) complex 8 (S)/
(R) 1:1
5.0
7.5
2.5
3.5
5.0 (1.00)
8.0 (1.07)
2.0 (0.80)
6.5 (1.86)
5.5
40.0
3.3
5.5
35.0
8.0
CONCLUSION
■
6.3
9.1
In conclusion, a set of Ru(II) and Ir(III) complexes with
different N-containing ligands based on a steroidal backbone
was synthesized and characterized by NMR spectroscopy and
X-ray crystallography. All evaluated complexes showed high
cytotoxicity in the tested cancer cell lines and were more active
in RT112 cP and MCF-7 cells than cisplatin. Remarkable is the
very low resistant factor of the complexes in the range between
0.33 and 1.22, indicating successful overcoming of the cisplatin
resistance. Especially promising results were obtained for the
complexes 7 and 10 with the steroidal ligand 5c, since the
advantageous high biocompatibility of 5c (IC50 > 50 μM) was
combined with a pronounced antiproliferative effect of the
complexes 7 (IC50 3 μM for RT112 and 1 μM for RT112 cP)
and 10 (IC502 μM for RT112 and 1 μM for RT112 cP) with
resistant factors 0.33 and 0.5, respectively. In breast cancer
cells, which show a proliferation dependency on hormone
expression, all complexes showed an effective cytotoxicity
including the complex 7 (IC50 = 2.5 μM). This antiproliferative
activity was significantly higher than for the well-established
ruthenium complexes RAPTA-T and NAMI-A, with IC50
values >200 μM and 800 μM (both MCF-7), reported by
Nazarov et al.46 and Pluim et al.,47 respectively. The cytotoxic
effect of the compounds was also higher than that of the
mentioned cholesterol ruthenium(III) complex incorporated
into a liposome bilayer, demonstrated by Simeone et al. (IC50
of about 70 μM),18 and was in the range of the testosterone−
ruthenium conjugate described by Lv et al. (IC50 of about 4.5
μM).27 Furthermore, the complexes showed a promising
selectivity for the cancer cells, exhibiting lower cytotoxicity
against normal fibroblasts compared to the tested cancer cell
lines.
6
7
8
9
10
11
12
13
14
15
(S-Ru) complex 8
(R-Ru) complex 8
Ir(III) complex 11
ligand 5b
Ru(II) complex 12
Ir(III) complex 13
ligand 5c
Ru(II) complex 7
Ir(III) complex 10
cisplatin
2.5
3.9
4.5
7.5
9.0
9.0
>50.0
3.0
2.0
3.5
5.5 (2.20)
6.0 (1.54)
2.5 (0.56)
7.0 (0.93)
7.5 (0.83)
11.0 (1.22)
>50.0
1.0 (0.33)
1.0 (0.50)
>50.0
6.0
5.5
5.7
9.0
7.0
8.5
6.6
2.5
8.5
22.5
10.5
7.0
6.9
>50.0
>50.0
>50.0
>50.0
5.5
17.5
>50.0
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) re-
agent can be reduced to blue-purple formazan by the
mitochondrial enzymes of living cells. The amount of resulting
formazan can be determined photometrically and correlates
directly with the cell viability, since this reaction can only take
place in metabolically active cells. The cytotoxicity was tested
in different tumor and somatic cells including bladder cancer
cells, which show resistance to cisplatin and breast cancer cells
(MCF7), which usually show hormone dependency. In order
to test a potential application in cisplatin resistant cells, the
human bladder carcinoma cell line RT112 and its cisplatin
resistant counterpart RT112 cP were cultivated with varying
concentrations (0.5−50 μM) of the ruthenium(II) (6, 7, 8,
and 12) and iridium(III) complexes (9, 10, 11, and 13), and
the cell viability was monitored after 72 h of incubation. In
addition, the cytotoxicity of the free ligands ((4-
bromophenyl)pyridine, 5a−5c) and cisplatin was evaluated
for comparison. There are some reports on the expression of
various steroid receptors in bladder cancer cells,44,45 assuming
that steroidal ligands could have an effect. All complexes
demonstrated higher cytotoxicity against RT112 cP cells
compared to cisplatin (IC50 values 1−11 μM and >50 μM,
respectively). The cytotoxicity of the complexes 8 (both (S)-
EXPERIMENTAL SECTION
■
Instrumental Measurements. ATR IR spectra were performed
on Bruker alpha-p and a FT-IR IFS 88 spectrometer. H and 13C
1
G
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NMR spectra were recorded on different types of Bruker Avance 400,
Bruker Avance III HD, or Bruker Avance 600 spectrometer with
residual proton signals of the deuterated solvent as the internal
standard. EI and FAB mass spectra (positive mode) were measured
on a Finnigan MAT95. Further information is given in the Supporting
Information.
General Procedure for Metallacyclization Reactions with
Ruthenium(II) and Iridium(III). Under an argon atmosphere, the
ligand (2.00 equiv), [RuCl2(p-cymene)]2 (1.00 equiv) or [IrCp*Cl2]2
(1.00 equiv), and KOAc (4.00 equiv) were dissolved in dry MeOH or
CH2Cl2 and stirred at room temperature for 24 h. The suspension was
concentrated, and the residue was purified by flash column
chromatography on silica gel to obtain the cyclometalated complexes
as yellow to orange solids. The reactions based on a steroidal ligand
were performed on a 30−120 μmol scale.
Crystal Structure Determinations. The single-crystal X-ray
diffraction study of 5b31 and 7 was carried out on a Bruker D8
Venture diffractometer with a Photon100 detector at 123(2) K using
Cu−Kα radiation (λ = 1.54178 Å). Direct Methods (SHELXS-97)48
was used for structure solution, and refinement was carried out using
SHELXL-2014 (full-matrix least-squares on F2).49 Hydrogen atoms
were localized by difference electron density determination and
refined using a riding model (H(O) free). Semiempirical absorption
corrections were applied. The absolute configuration was determined
by refinement of Parsons’ x-parameter.50 The single-crystal X-ray
diffraction study of (R-Ru)-8, 12, and 13 was performed on a Stoe
StadiVari diffractometer using Ga Kα radiation (λ = 1.34143 Å)
generated by an Metaljet X-ray source. The crystals were kept at
180.15 K during data collection. Using Olex2,51 the structures were
solved with the ShelXS48 structure solution program using Direct
Methods and refined with the ShelXL49 refinement package using
Least Squares minimization. Non-hydrogen atoms were refined with
anisotropic displacement parameters; hydrogen atoms were modeled
on idealized positions.
CCDC 1521243 (5b), 1859054 (7), 1944097 ((R-Ru)-8),
1944098 (12), and 1944099 (13) contain the supplementary
crystallographic data for this paper. These data can be obtained free
of charge from the Cambridge Crystallographic Data Centre via www.
ccdc.cam.ac.uk/data_request/cif.
Computational Details. Structure optimizations were done on
the BP8635,36/def2-TZVPP37 level of theory using the TURBO-
MOLE 7.1 program package.52 Solvent effects of chloroform were
taken into account with the COSMO solvation model.53 The RI-
approximation was used throughout.54 Stationary points were verified
to be minimum energy structures by numerically calculating the
molecular Hessian and analyzing the so obtained vibrational
frequencies. The numerical frequencies were used to calculate
thermodynamic properties at 298.15 K and 1 bar in harmonic and
ideal gas approximations. NMR chemical shifts were calculated on the
basis of Gauge Including Atomic Orbitals (GIAO).55
Cell Culture. RT112 (human bladder carcinoma cell line), RT112
cP (cisplatin-resistant), NHDF (normal human dermal fibroblasts),
and MCF-7 (breast cancer cell line) were cultured in RPMI (Roswell
Park Memorial Istitute) medium (Gibco, for RT112 and RT112 cP)
or DMEM (Dulbecco’s modified eagle medium, Gibco, for NHDF
and MCF-7) supplemented with 10% fetal bovine serum (FBS,
Gibco) and 1% penicillin/streptavidin (Gibco) at 37 °C, 5% CO2, and
a humid atmosphere. For all in vitro experiments, cells were
trypsinized (0.05% trypsin-EDTA, Gibco) and seeded in 96-well-
plates (toxicity assay) at the required densities. Incubation was
performed under the culture conditions as described above.
Cytotoxicity Assay. RT112 (human bladder carcinoma cell line),
RT112 cP (cisplatin-resistant), NHDF (normal human dermal
fibroblasts), and MCF-7 (breast cancer cell line) were seeded in the
96-well-plates at a density of 1 × 104 cells/well in RPMI (RT112 and
RT112 cP) medium or DMEM (NHDF and MCF-7) supplemented
with 10% FCS and 1% penicillin/streptomycin. After 24 h of
incubation at 37 °C and 5% CO2, the medium was removed and the
cells were treated with various concentrations of the compounds in
the corresponding culture medium and incubated for 72 h at 37 °C
and 5% CO2. The stock solutions of the compounds were prepared in
DMSO and the end concentration of the solvent in the test dilutions
was held under 0.5%. Since the cytotoxic activity of cisplatin was
shown to be affected by DMSO,56 a stable 5 mM stock solution of
cisplatin in DPBS−/− (Gibco) was prepared. As a negative control, the
cell culture medium was exchanged without addition of the
compounds. Thereafter, 15 μL of the MTT reagent (Promega) was
given in each well. For the positive control, Triton X-100 (1%) was
added in some wells before treating them with the MTT reagent.
After 3 h of incubation, the cells were lysed using the Stop Solution
(Promega) to release the blue-purple formazan. The cell viability was
determined by measuring the absorbance of the resulting formazan at
595 nm using a multiwell plate reader (SpectraMax ID3, Molecular
Devices, USA) and calculated in relation to the negative control.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.9b02402.
■
S
Additional spectroscopic data, experimental and compu-
tational details, and characterization data (PDF)
Accession Codes
CCDC 1859054 and 1944097−1944099 contain the supple-
mentary crystallographic data for this paper. These data can be
obtained free of charge via www.ccdc.cam.ac.uk/data_request/
cif, or by emailing data_request@ccdc.cam.ac.uk, or by
contacting The Cambridge Crystallographic Data Centre, 12
Union Road, Cambridge CB2 1EZ, UK; fax: +44 1223 336033.
AUTHOR INFORMATION
Corresponding Author
*E-mail: braese@kit.edu.
■
ORCID
Vanessa Koch: 0000-0002-2115-3124
̈
Stefan Brase: 0000-0003-4845-3191
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
■
We acknowledge the DFG Core Facility MOLECULE
ARCHIVE (grant numbers: BR1750/40-1, JU2909/5-1) for
the management and provision of the compounds for
screening. V.K. gratefully acknowledges the Studienstiftung
des Deutschen Volkes and W.F., the Carl-Zeiss-Stiftung for
financial support. The work was supported by the Deutsche
Forschungsgemeinschaft (DFG), within the Research Training
Group 2039 (A.M., U.S. S.B.) and the Helmholtz Program
Biointerfaces in Technology and Medicine (BIFTM; U.S.,
A.M., S.B.). We thank Prof. Dr. Frank Breher for supporting
̈
this cooperation, Dr. Beate Koberle for providing cell lines, and
Magdalena Winklhofer for assistance in in vitro experiments.
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