Biological activity of neutral and cationic iridium(III) complexes with κp and κp,κS coordinated Ph2PCH2S(O) xPh (x = 0-2) ligands

Article abstract of DOI:10.1016/j.ejmech.2013.08.025

Neutral iridium(III) complexes of the type [Ir(η⁵-C ₅Me₅)Cl₂{Ph₂PCH₂S(O) _xPh-κP}] (1-3) with diphenylphosphino-functionalized methyl phenyl sulfides, sulfoxides, and sulfones Ph₂PCH₂S(O) _xPh (x = 0, L1; 1, L2; 2, L3) and the cationic complex [Ir(η⁵-C₅Me₅)Cl{Ph₂PCH ₂SPh-κP_,κS}][PF₆] (4) were synthesized and fully characterized analytically and spectroscopically. Furthermore, the structure of 2 was determined by X-ray diffraction analysis. The biological potential of the neutral and cationic iridium(III) complexes was tested in vitro against the cell lines 8505C, A253, MCF-7, SW480 and 518A2. Complex [Ir(η⁵-C₅Me₅)Cl₂{Ph ₂PCH₂S(O)Ph-κP}] (2), with ligand L2 κP coordinated containing a pendent sulfinyl group, is the most active one (IC ₅₀ values of about 3 μM), thus, with activities comparable to cisplatin. Complex 2 proved to have an even a higher antiproliferative activity than cisplatin against 8505C and SW480 cell lines, used as a model system of highly anaplastic cancers with low sensitivity to conventional chemotherapeutics such as cisplatin. Additional experiments demonstrated that apoptosis and autophagic cell death contribute to the drug's tumoricidal action.

Full text of DOI:10.1016/j.ejmech.2013.08.025

European Journal of Medicinal Chemistry 69 (2013) 216e222
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Biological activity of neutral and cationic iridium(III) complexes with
k
P and
k
P,
k
S coordinated Ph2PCH2S(O)xPh (x ¼ 0e2) ligands
a
ꢀ b
ꢀ b
ꢀ b
ꢀ b
Gerd Ludwig , Sanja Mijatovic , Ivan RanCelovic , Mirna Bulatovic , Djordje Miljkovic ,
Danijela Maksimovic-Ivanic , Marcus Korb , Heinrich Lang , Dirk Steinborn ,
Goran N. KaluCerovic
c
c
a
ꢀ
ꢀ a
ꢀ b
,
*
a Institute of Chemistry, Martin Luther University Halle-Wittenberg, Kurt-Mothes-Straße 2, D-06120 Halle, Germany
b Institute for Biological Research “Sinisa Stankovic”, University of Belgrade, Bulevar despota Stefana 142, 11060 Belgrade, Serbia
c Institute of Chemistry, Chemnitz University of Technology, Straße der Nationen 62, D-09111 Chemnitz, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Neutral iridium(III) complexes of the type [Ir(
diphenylphosphino-functionalized methyl phenyl sulfides, sulfoxides, and sulfones Ph2PCH2S(O)xPh
h
5-C5Me5)Cl2{Ph2PCH2S(O)xPh-
k
P}] (1ꢀ3) with
Received 8 May 2013
Received in revised form
12 August 2013
Accepted 14 August 2013
Available online 29 August 2013
(x ¼ 0, L1; 1, L2; 2, L3) and the cationic complex [Ir(
h
5-C5Me5)Cl{Ph2PCH2SPh-
kP,kS}][PF6] (4) were
synthesized and fully characterized analytically and spectroscopically. Furthermore, the structure of 2
was determined by X-ray diffraction analysis. The biological potential of the neutral and cationic iri-
dium(III) complexes was tested in vitro against the cell lines 8505C, A253, MCF-7, SW480 and 518A2.
Complex [Ir(
h
5-C5Me5)Cl2{Ph2PCH2S(O)Ph-
k
P}] (2), with ligand L2
kP coordinated containing a pendent
Keywords:
sulfinyl group, is the most active one (IC50 values of about 3
m
M), thus, with activities comparable to
Iridium(III) complexes
Sulfur-functionalized phosphorus ligands
Cytotoxic activity
cisplatin. Complex 2 proved to have an even a higher antiproliferative activity than cisplatin against
8505C and SW480 cell lines, used as a model system of highly anaplastic cancers with low sensitivity to
conventional chemotherapeutics such as cisplatin. Additional experiments demonstrated that apoptosis
and autophagic cell death contribute to the drug’s tumoricidal action.
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
hand, only a few steps have been taken in the field of iridium-based
anticancer agents [18e25]. This can be, at least in part, attributed to
Bioinorganic chemistry is a fast-growing area of fascinating
opportunities and creative power [1]. This is reflected, among
others, in the discovery of the cancerostatic activity of cisplatin by
Rosenberg in 1965 [2,3]. Cisplatin itself is still used and is consid-
ered a benchmark for the entire field of metal-based anticancer
drugs that have since been developed [4]. Due to the disadvantage
of the dose-dependent side effects of cisplatin as well as the
resistance of some carcinomas, it is understandable that a wide
range of novel transition metal complexes were screened for their
use as therapeutic agents [5e10]. Thus, first promising steps were
taken in the field of titanium-, ruthenium- and gold-based anti-
cancer agents [11]. For instance, the octahedral ruthenium(III)
the relatively high kinetic inertness of iridium(III) complexes due to
their low-spin d6 valence electron configuration [26]. On this basis,
for example, the absence of cytotoxic activity of the iridium(III)
complexes [imiH]trans-[Ir(imi-kN)(dmso-kS)Cl4] and [indH]trans-
[Ir(ind- N)2Cl4] has been explained [27]. Despite these initial set-
k
backs, iridium(III) complexes such as fac-[Ir(NXN)(dmso-
kS)Cl3]
(NXN ¼ diimine type ligand) I (Fig. 1) [28], with a promising anti-
cancer activity have been prepared. On a broad scale h
5-pentam-
ethylcyclopentadienyl iridium(III) complexes of the type [Ir(h5
-
C5Me5)Cl(XXY)]nþ (XXY ¼ bidentate ligand) have been tested for
their ability to decrease the cell viability. Originally, compounds
with N,N’ ligands (XXY ¼ ethylendiamine, 2,20-bipyridine, 1,10-
complexes [imiH]trans-[Ru(imi-
azole) and [indH]trans-[Ru(ind-
reached clinical trials for cancer treatment [12e17]. On the other
k
k
N)(dmso-
N)2Cl4] (ind ¼ indazole) have
k
S)Cl4] (imi
¼
imid-
phenanthroline) [29] as well as [Ir(
h
5-C5Me5)Cl2(PTA)] (PTA ¼
1,3,5-triaza-7-phosphaadamantane) [23] were found to have no
cytotoxicity against several cancer cell lines. On the other hand,
Sheldrick et al. [30] and Sadler et al. [29] found that complexes of
the above mentioned type bearing a functionalized bipyridine or
phenanthroline ligand and a functionalized C5Me5 ligand, respec-
tively, as shown in the examples II and III (Fig. 1), exhibited high
* Corresponding author. Tel.: þ49 345 5525678; fax: þ49 345 5527028.
ꢀ
E-mail address: goran.kaluderovic@chemie.uni-halle.de (G.N. KaluCerovic).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.08.025

G. Ludwig et al. / European Journal of Medicinal Chemistry 69 (2013) 216e222
217
Fig. 1. Examples of iridium-based anticancer drugs.
biological potential. Furthermore, complex IV (Fig. 1) bearing an
anionic NXC ligand, instead of a neutral diimine type ligand (such
as bpy), possess a promising anticancer ability with IC50 value
(IC50 ¼ concentration of compound that inhibits 50% of cell growth)
shift of the phosphorus resonance by ꢀ14 ppm. Whereas in the
1
uncoordinated ligands the magnitude of the JP,C coupling con-
stants of the a-carbon atoms are in direct relation to the oxidation
state of the sulfur atom (1JP,C ¼ 23.2 Hz (L1) < 31.2 Hz (L2) < 35.7 Hz
of 0.7
mM (cell line A2780), which is comparable to that of cisplatin
(L3)), the coordination to Ir(III) aligns all the 1JP,C coupling constants
(IC50 ¼ 1.2
mM) [31,32].
at about 26 Hz. The resonances of the h
5-C5Me5 ligand in com-
To the best of our knowledge, no cytotoxically active iridium(III)
complexes with phosphorus ligands have been reported so far.
Previously, our group has reported on neutral and cationic ruth-
plexes 1ꢀ3 are in a narrow range and, thus, they are not dependent
on the pendent S(O)xPh group. Even the values of the cationic
complex 4 differ only marginally (Table 1).
enium(II) complexes with
u-diphenylphosphino-functionalized
Crystals of [Ir(h kP}] (2) suitable for
5-C5Me5)Cl2{Ph2PCH2S(O)Ph-
alkyl phenyl sulfides, sulfoxides and sulfones that exhibit promising
anticancer activity, partially by one order of magnitude higher in
activity than cisplatin [33,34]. Here, we report on the biological
X-ray diffraction analysis were obtained from solutions of methy-
lene chloride/n-pentane at room temperature. The compound
crystallized as isolated molecules without unusual intermolecular
interactions (shortest distance between non-hydrogen atoms:
potential of iridium(III) complexes of the type [Ir(
h
5-C5Me5)
P, S}]
5-C5Me5)Cl{Ph2PCH2SPh-
k
k
3.370(3) A, C12/C28 ). The molecular structure is shown in Fig. 2
and selected structural parameters are given in the figure caption.
The iridium(III) complex adopts a typical half sandwich (“three-
legged piano stool”) structure, in which the metal center is coor-
0
ꢁ
Cl2{Ph2PCH2S(O)xPh- P}] and [Ir(
k
h
[PF6], especially on a correlation between the oxidation state of the
sulfur atom (x ¼ 0e2) and the cytotoxic activity as well as on the
tumoricidal action of these anticancer drugs.
dinated to a h5-C5Me5 ligand (distance Ir to ring centroid 1.835 A),
ꢁ
two chlorido ligands as well as the Ph2PCH2S(O)Ph-kP (L2) ligand.
2. Results and discussion
The angles at the iridium(III) atom are close to 90ꢁ (86.7(1)e
89.8(2)ꢁ), so that the structure can be viewed as a distorted octa-
2.1. Syntheses and characterization
ꢁ
hedron. The IreCl bond lengths (2.407(3)/2.401(3) A) as also the Ire
ꢁ
P bond length (2.310(3) A) are in the expected range (median IreCl:
Mononuclear neutral iridium(III) complexes of the type [Ir(h5
C5Me5)Cl2{Ph2PCH2S(O)xPh-
P}] (1ꢀ3) were synthesized through
reactions of the dinuclear complex [{Ir(
5-C5Me5)Cl2)}2] with
diphenylphosphino-functionalized methyl phenyl sulfides, sulfox-
ides, and sulfones Ph2PCH2S(O)xPh (x ¼ 0, L1; 1, L2; 2, L3) in
methylene chloride (Scheme 1, reaction pathway a). As reported for
complex 1 [35] and also for other cases [36], the donicity of the P
atom is high enough to cleave the IreCleIr bridges. Furthermore,
-
ꢁ
ꢁ
2.419 A, lower/higher quartile: 2.315/2.606 A, n ¼ 543; median Ire
k
ꢁ
ꢁ
P: 2.316 A, lower/higher quartile: 2.151/2.462 A, n ¼ 543; n e
h
number of observations [37]).
2.2. Biological studies
To evaluate the cytotoxic potency of the iridium(III) complexes
1e4, 8505C human thyroid carcinoma, A253 submandibular car-
cinoma, MCF-7 breast adenocarcinoma, SW480 colon adenocarci-
noma, and 518A2 melanoma cell lines were exposed to a wide
range of doses of these compounds. Cell viability was estimated
after 96 h by SRB assay [38]. According to the obtained results all
complexes expressed a dose-dependent decrease of cell viability
(Fig. 3). The IC50 values of iridium(III) complexes 1ꢀ4 are listed in
Table 2; the activities of cisplatin are included for comparison. For
further understanding the IC50 values of the corresponding ligands
L1eL3 and the related ruthenium complexes are given in Table S1.
Generally, the phosphineesulfide L1 and the phosphineesulf-
oxide L2 possess an up to forty times lower in vitro activity than the
related iridium(III) complexes. The activities of the phosphinee
sulfone L3 are similar and in two cases (cell lines 518A2 and 8505C)
even three times better than those of the corresponding iridium(III)
complex 3. The only complex with activities comparable to cisplatin
the cationic iridium(III) complex [Ir(
,kS}][PF6] (4) was obtained through chlorido abstraction with
h
5-C5Me5)Cl{Ph2PCH2SPh-
k
P
[NH4][PF6] starting from the corresponding neutral iridium(III)
complex 1 (Scheme 1, reaction pathway b). The complexes 1ꢀ3
were obtained as orange powders in yields between 64 and 71% and
the cationic complex 4 as reddish powder in a yield of 77%. Both,
the neutral and the cationic complexes 1ꢀ4 are stable in air over
weeks and soluble in methylene chloride and dimethyl sulfoxide.
The identities of complexes 1e4 were confirmed by elemental an-
alyses, NMR spectroscopy (1H, 13C, 31P), and X-ray single-crystal
structure analysis (2).
All 1H, 13C, and 31P NMR spectroscopic parameters of complexes
1ꢀ4 are in the expected range; selected values are given in Table 1
[35]. Coordination-induced shifts (CIS) of the
a-carbon atoms
(
dC(coord.)
between ꢀ2.9 and ꢀ3.6 ppm. Furthermore,
ligands to Ir(III) results in CIS of the phosphorus resonances
dP(coord.) dP(uncoord.)) between 20 and 24 ppm. In contrast, the
additional S coordination of L1 in complex 4 results in a high-field
ꢀ
dC(uncoord.)) of the ligands Ph2PCH2S(O)xPh were found
k
P coordination of the
is [Ir(
ordinated containing a pendent sulfinyl group (IC50 values of about
M). Thus, 2 exhibited slightly higher antiproliferative activity
h kP}] (2) with ligand L2 kP co-
5-C5Me5)Cl2{Ph2PCH2S(O)Ph-
(
ꢀ
3
m

218
G. Ludwig et al. / European Journal of Medicinal Chemistry 69 (2013) 216e222
Scheme 1. Synthetic routes to iridium(III) complexes bearing Ph2PCH2S(O)xPh-kP ligands (1e3) and the Ph2PCH2SPh-kP,kS ligand (4), respectively.
than cisplatin on the cisplatin resistant cell lines 8505C and SW480,
whereas on two cisplatin sensitive cell lines (518A2 and A253) 2
was found twice less efficient than cisplatin. It is worth mentioning
that the iridium(III) complex 2 exhibited higher activities (up to one
order of magnitude) on all tested cell lines than the related ruth-
exposed to 2 massive autophagic vesicles were determined indi-
cating an enhanced autophagic process (Fig. 4D). Keeping in mind
that autophagy serves to save the cells from damage, but can
mediate cell death in some circumstances, cells were exposed to
specific autophagic inhibitor 3-MA concomitantly with 2. The
measurement of the cell viability revealed a 10% increase in a
number of tumor cells in cultures exposed to both 2 and the in-
hibitor in comparison to cultures exposed to the drug alone
(Fig. 4E). This result confirms that besides apoptosis, autophagic
cell death also contributes to the drugs tumoricidal action.
In an attempt to explore the involvement of oxidative stress in
the cytotoxicity of 2, cells were stained with the specific dye DHR
(dihydrorhodamine 123) which became fluorescent in contact with
reactive oxygen (ROS) and nitrogen species (RNS). The cumulative
effect was measured after 24 and 48 h of exposure to 2. Interest-
ingly, instead of enhanced ROS and RNS levels, significantly
decreased levels of free radicals were determined (Fig. 4F). The
observed phenomenon was previously described in thyroid cancer
enium(II) complex [Ru(h kP}] [33].
6-p-cym)Cl2{Ph2PCH2S(O)Ph-
This result is in contrast to the iridium(III) complexes 1, 3, and 4
with the sulfide- (L1) and sulfonyl-functionalized ligand (L3), that
exhibited a comparable or even less ability to decrease the cell
viability in comparison with the analogous ruthenium(II) com-
plexes
{Ph2PCH2SPh-
[Ir(
5-C5Me5)Cl2{Ph2PCH2SPh-
cationic complex [Ir(
5-C5Me5)Cl{Ph2PCH2SPh-
containing the Ph2PCH2SPh ligand P and kP,k
[Ru(
h
k
6-p-cym)Cl2{Ph2PCH2S(O)xPh-
P,
k
P}]/[Ru(
S}][PF6] (x ¼ 0, 2) [33,34]. The neutral complex
P}] (1) and the corresponding
P, S}][PF6] (4),
S coordinated,
h
6-p-cym)Cl
k
h
k
h
k k
k
respectively, exhibited similar antitumor activities against investi-
gated cell lines, with the exception of the 518A2 cell line. These
findings are in contrast to the analogous ruthenium(II) complexes
[Ru(
Cl2{Ph2PCH2SPh-
h k kS}][PF6] and [Ru(h
6-p-cym)Cl{Ph2PCH2SPh- 6-p-cym)
P,
P}], where the cationic complex exhibited an up
k
to sixteen times higher inhibition than the neutral one against
three cell lines (8505C, MCF-7, SW80) [34].
The sensitivity of the cancer cells under investigation (although
derived from different origins and types of tumors) on [Ir(h5
-
C5Me5)Cl2{Ph2PCH2S(O)Ph-kP}] (2), the most potent complex, was
similar. Because of low level of differentiation and dominant stem
phenotype, 8505C thyroid carcinoma was selected for further
analysis as a model of highly malignant and almost incurable type
of the tumor. Thus, to explore the mechanism of action, 8505C cells
were treated with the IC50 dose of complex 2 for 48 h. Then, the
presence of early and late apoptotic cells were analyzed with
double Ann/PI staining. As seen in Fig. 4A, in comparison to the
control (non-treated cells), a 48 h treatment with 2 gave rise to an
increase of both early apoptotic cells marked by externalization of
phosphatidylserin and late apoptotic/necrotic cells stained by both
Ann and PI. In parallel, 22% of cells displayed enhanced activation of
caspases confirming that caspase-dependent apoptosis is probably
behind the action of this compound (Fig. 4B). Accordingly, cell cycle
analysis showed an increased percentage of hipodiploid cells with
fragmented DNA upon treatment with 2 pointing out once more
that apoptosis is the dominant mode of action of this kind of
iridium-based drugs (Fig. 4C). In parallel, in the cytoplasm of cells
Table 1
Selected NMR spectroscopic data
Cl2{Ph2PCH2S(O)xPh- P}] (1e3) and [Ir(
(
h
d
in ppm,
J
in Hz) of [Ir(
h
5-C5Me5)
kP,kS}][PF6] (4).
k
5-C5Me5)Cl2{Ph2PCH2SPh-
xa
Ph2PCaH2S(O)xPh
C5Me5 (ring)
da-CH (1JP,H
)
da-C (1JP,C
)
dP
dH (4JP,H
)
dCMe (3JP,C dC (2JP,C
) )
Fig. 2. Molecular structure of [Ir(h kP}] in crystals of 2.
5-C5Me5)Cl2{Ph2PCH2S(O)Ph-
The ellipsoids are shown with a probability of 30%. From the disordered PhS(O)CH2
group only the major occupied position is shown. H atoms have been omitted for
1
2
3
4
0 (
1 (
2 (
0 (
k
k
k
k
P)
P)
P)
4.29 (4.8)
29.1 (26.8)
2.3 1.26 (2.3) 8.2 (1.0)
92.2 (2.8)
92.5 (3.0)
92.5
4.04e4.57 55.9 (25.4) ꢀ4.8 1.37 (2.4) 8.0 (1.0)
clarity. Selected structural parameters (distances in A, angles in ꢁ; values for the
ꢁ
3.96e4.01 56.5 (26.0) ꢀ5.2 1.33 (2.4) 8.3
disordered group (63/37%) are given separated by a slash): Ir1eCl1 2.407(3), Ir1eCl2
2.401(3), Ir1eP1 2.310(3), Cl1eIr1eCl2 89.8(2), Cl1eIreP1 86.7(1), Cl2eIreP1 87.7(1),
C11eS1eC17 93.3(1)/103(2), O1eS1eC17 115.5(2)/98(3), O1eS1eC11 102.7(1)/120(2).
P,
k
S) 5.12e5.18 29.6
ꢀ31.9 1.73 (2.9) 8.7 (1.0)
92.3
a
The coordination mode of the ligand is given in parentheses.

G. Ludwig et al. / European Journal of Medicinal Chemistry 69 (2013) 216e222
219
Fig. 3. Representative graphs showing survival (in %) of cells grown for 96 h in the presence of increasing concentrations of compounds 1e4.
2) The oxidation state of the sulfur (eSPh vs. ꢀS(O)Ph vs. e
S(O)2Ph) in the ligands is directly correlated to the cytotoxic
activity. Thus, complex 2 with the pendent sulfinyl exhibited
the highest biological potential, followed by complexes 1 and 3
with the pendent sulfide and sulfonyl group, respectively, as
demonstrated in Fig. 5.
cells treated with proteasome inhibitor (bortezomib) and explained
with cell-specific response to toxic stimuli [39,40]. Scavenging of
ROS by thyroid cancer cells in response to cytotoxic stimuli could at
least in part be assigned to elevated redox protection. However, this
protection of the cells against oxidative stress did not compromise
the effectiveness of the drug.
3) In the case of the Ph2PCH2SPh ligand (L1) the coordination
mode (kP vs. kP,kS), which goes along with a transition from a
2.3. Conclusion
neutral to a cationic complex (1 vs. 4), has no significant in-
fluence on the cytotoxic activity of the iridium(III) complexes
(cf. Fig. 5).
In this work, neutral and cationic iridium(III) complexes of the
type [Ir( P}] (x ¼ 0, 1; 1, 2; 2, 3) and
h
5-C5Me5)Cl2{Ph2PCH2S(O)xPh-
k
4) The iridium(III) complex 2 with a sulfinyl functionalized-ligand
possesses the highest potential to decrease the cell viability
[Ir( kP,kS}][PF6] (4), with bound
h
5-C5Me5)Cl{Ph2PCH2SPh-
diphenylphosphino-functionalized methyl phenyl sulfide, sulfoxide
and sulfone ligands, were synthesized and fully characterized. Their
biological potential was tested and proven against several cell lines.
The following conclusions can be drawn:
with an IC50 value of 1.9 mM against the A253 cell line.
5) The iridium(III) complexes 1, 3, and 4 with a sulfide- and
sulfonyl-functionalized ligand showed comparable or even less
cytotoxic activity in comparison with the related ruthenium(II)
complexes [Ru(
cym)Cl{Ph2PCH2SPh-
values given in Table 2 and Supplementary material S1). In
contrast, the iridium(III) complex with the sulfinyl-
h
6-p-cym)Cl2{Ph2PCH2S(O)xPh-
P,
k
P}]/[Ru(
h
6-p-
1) In vitro tests revealed that the cytotoxic potency of the sulfur-
k
k
S}][PF6] (x ¼ 0, 2; see Fig. 5 and the
functionalized phosphanes Ph2PCH2S(O)xPh is strongly
increased by coordination to the Ir(
moiety. Thus, the cytotoxic activities were found up to forty
times higher than those of the corresponding ligands, cf. the
values given in Table 2 with those in Table S1.
h
5-C5Me5)Clx (x ¼ 1, 2)
2
functionalized ligand was found up to ten times more active
than the corresponding ruthenium(II) complex.
Table 2
IC50 valuesa
(
m
M) of iridium(III) complexes 1ꢀ4 in comparison with cisplatin.
Compound
xb
518A2
8505C
A253
MCF-7
SW480
1
2
3
4
0 (
1 (
2 (
0 (
k
k
k
k
P)
P)
P)
13.6 ꢂ 2.3
2.4 ꢂ 0.1
18.1 ꢂ 1.6
9.8 ꢂ 0.5
1.5 ꢂ 0.2
15.4 ꢂ 1.4
3.5 ꢂ 0.5
36.8 ꢂ 5.7
16.8 ꢂ 3.5
5.0 ꢂ 0.2
10.6 ꢂ 1.3
1.9 ꢂ 0.2
34.6 ꢂ 1.1
10.2 ꢂ 2.8
0.8 ꢂ 0.1
18.5 ꢂ 3.2
3.2 ꢂ 0.4
42.0 ꢂ 7.3
16.0 ꢂ 1.9
2.0 ꢂ 0.1
13.4 ꢂ 3.0
2.3 ꢂ 0.2
28.5 ꢂ 11.7
14.6 ꢂ 3.4
3.2 ꢂ 0.2
P,kS)
Cisplatin
a
Mean values ꢂ SD (standard deviation) from three experiments.
b
The coordination mode of the ligand is given in parentheses.

220
G. Ludwig et al. / European Journal of Medicinal Chemistry 69 (2013) 216e222
Fig. 4. The effect of [Ir(h kP}] (2) on malignant cell death. 8505C cells were exposed to IC50 doses of compound 2 and (A) apoptosis, (B) caspase
5-C5Me5)Cl2{Ph2PCH2S(O)Ph-
activation, (C) cell cycle distribution, (D) presence of autophagic vesicles, (E) cell viability in presence of autophagic inhibitor (3-MA) or (F) production of ROS and RNS were
evaluated.
In conclusion, in iridium(III) complexes of the type [Ir(h5
C5Me5)Cl2{Ph2PCH2S(O)xPh-
P}] (x ¼ 0e2; 1e3) a correlation of the
-
yielding an orange powder which was filtered off, washed with n-
pentane (3 ꢃ 2 mL), and dried under vacuum.
k
oxidation state of the sulfur atom at the pendent group to a
decrease of the cell viability was observed, whereas complex 2
(x ¼ 1) proved to be the most active against the 8505C and SW480
cell lines which display a low sensitivity to cisplatin. Additional
experiments on cell line 8505C confirmed that apoptosis and
autophagic cell death contribute to drug’s tumoricidal action and
that 2 significantly decreased the level of free radicals.
1 (x ¼ 0). Yield: 153 mg (68%). HRMS (ESI): m/z Calc. for
[C29H32ClIrPS]þ: 669.1251; found for [M ꢀ Cl]þ: 669.1243. Anal.
Found: C, 49.01; H, 4.23. Calcd for C29H32Cl2IrPS (706.73): C, 49.29;
H, 4.56. 1H NMR (400 MHz, CD2Cl2):
d
1.26 (d, 4JP,H ¼ 2.31 Hz, 15H,
CH3), 4.29 (d, 2H, 2JP,H ¼ 4.75 Hz, CH2), 6.85e7.87 (m, 15H, HPh). 13
C
3
NMR (100 MHz, CD2Cl2):
d
8.2 (d, JP,C ¼ 1.0 Hz, CCH3), 29.1 (d,
1JP,C ¼ 26.8 Hz, CH2PPh2), 92.2 (d, 2JP,C ¼ 2.8 Hz, CCH3), 123.8e135.3
(CPh). 31P NMR (162 MHz, CD2Cl2):
d 2.3 (s).
2 (x ¼ 1). Yield: 164 mg (71%). HRMS (ESI): m/z Calc. for
[C29H32ClIrOPS]þ: 685.1200; found for [M ꢀ Cl]þ: 685.1193. Anal.
Found: C, 47.89; H, 4.17. Calcd for C29H32Cl2IrOPS (722.73): C, 48.19;
3. Experimental
H, 4.46. 1H NMR (400 MHz, CD2Cl2):
d
1,37 (d, 4JP,H ¼ 2.41 Hz, 15H,
3.1. General comments
CH3), 4.04e4.57 (m, 2H, CH2), 7.54e8.09 (m, 15H, HPh). 13C NMR
3
All reactions and manipulations were carried out under argon
using standard Schlenk techniques. Solvents were dried (n-pentane
over Na/benzophenone; methanol over magnesium; methylene
chloride over CaH2) and freshly distilled prior to use. NMR spectra
(1H, 13C, 31P) were recorded at 27 ꢁC on Varian Gemini 200 and VXR
400 spectrometers. Chemical shifts are relative to solvent signals
(CD2Cl2, dH 5.32, dC 53.8) as internal references; dP is relative to
external H3PO4 (85%). Microanalyses (C, H) were performed in the
Microanalytical Laboratory of the University of Halle using a CHNS-
932 (LECO) elemental analyzer. High-resolution ESI mass spectra
were obtained from a Bruker Apex III Fourier transform ion cyclo-
tron resonance (FTR-ICR) mass spectrometer (Bruker Daltonics)
equipped with an Infinity cell, a 7.0 T superconducting magnet
(Bruker), an rf-only hexapole ion guide, and an external APOLLO
electrospray ion source (Agilent, off-axis spray). The sample solu-
tions were introduced continuously via a syringe pump with a flow
(100 MHz, CD2Cl2):
d
8.0 (d, JP,C ¼ 1.0 Hz, CCH3), 55.9 (d,
1JP,C ¼ 25.4 Hz, CH2PPh2), 92.5 (d, 2JP,C ¼ 3.0 Hz, CCH3), 123.8e135.3
(CPh). 31P NMR (162 MHz, CD2Cl2):
d
ꢀ4.8 (s).
3 (x ¼ 2). Yield: 151 mg (64%). HRMS (ESI): m/z Calc. for
[C29H32ClIrO2PS]þ: 701.1149; found for [M ꢀ Cl]þ: 701.1140. Anal.
Found: C, 47.34; H, 4.51. Calcd for C29H32Cl2IrO2PS (738.72): C,
47.15; H, 4.37. 1H NMR (400 MHz, CD2Cl2):
d
1.33 (d, 4JP,H ¼ 2.39 Hz,
15H, CH3), 3.96e4.01 (d, 1H, CH2), 4.61e4.66 (d, 1H, CH2), 7.31e8.08
(m, 15H, HPh). 13C NMR (100 MHz, CD2Cl2):
d 8.3 (s, CCH3), 56.5 (d,
1JP,C ¼ 26.0 Hz, CH2PPh2), 92.5 (s, CCH3), 124.0e135.5 (CPh). 31P NMR
(162 MHz, CD2Cl2):
d
ꢀ5.2 (s).
3.3. Preparation of [IrCl(h kP,kS}][PF6] (4)
5-C5Me5){Ph2PCH2SPh-
The respective ligand (0.32 mmol) was added, with stirring, to a
methanol solution (30 mL) of [{IrCl2(
5-C5Me5)}2] (127 mg,
rate of 120
prepared according to literature procedures [41,42].
m h
L $ hꢀ1. [{IrCl2( 5-C5Me5)}2] and the ligands were
h
0.16 mmol) and the solution was heated under reflux for 3 h.
Subsequently, [NH4][PF6] (6 equiv.) was added. After storage
at ꢀ70 ꢁC overnight, the precipitate obtained was filtered off,
washed with diethyl ether (3 ꢃ 2 mL), and dried under vacuum.
Yield: 201 mg (77%).
3.2. Preparation of [IrCl2(h kP}] (1e3)
5-C5Me5){Ph2PCH2S(O)xPh-
The respective ligand (0.32 mmol) was added, with stirring to a
methylene chloride solution (25 mL) of [{IrCl2(
5-C5Me5)}2]
(127 mg, 0.16 mmol). The solution was stirred at r.t. overnight
HRMS (ESI): m/z Calc. for [C29H32ClIrPS]þ: 669.1251; found for
h
[M]þ: 669.1258. Anal. Found: C, 42.93; H, 4.12. Calcd for
C
29H32ClF6IrP2S (816.24): C, 42.67; H, 3.95. 1H NMR (400 MHz,

G. Ludwig et al. / European Journal of Medicinal Chemistry 69 (2013) 216e222
221
Fig. 5. Range of the cytotoxic activity (based on the IC50 values for the five cell lines under investigation) of the iridium(III) complexes 1ꢀ4 ([Ir] ¼ Ir(
h
5-C5Me5)Clx; x ¼ 1, 2) and of
the related ruthenium(II) complexes [Ru(
h
6-p-cym)Cl2{Ph2PCH2S(O)xPh-
k
P}] ([Ru] ¼ Ru(
h
6-p-cym)Cl2). For comparison, the corresponding values of cisplatin and the uncoordinated
ligands Ph2PCH2S(O)xPh L1eL3 (dashed lines) are given. aFour values; exception cell line MCF-7 (IC50 ¼ 115.0
m
M). bFour values; exception cell line 518A2 (IC50 ¼ 5.5
mM).
CD2Cl2):
d
1.73 (d, 4JP,H ¼ 2.91 Hz, 15H, CH3), 5.12e5.18 (m, 1H, CH2),
of charge from the Cambridge Crystallographic Data Centre via
www.ccdc.cam.ac.uk/data_request/cif.
7.20e7.25 (m, 1H, CH2), 7.20e7.71 (m, 15H, HPh). 13C NMR (100 MHz,
3
CD2Cl2):
d
8.7 (d, JP,C ¼ 1.0 Hz, CCH3), 29.6 (s, CH2PPh2), 92.3 (s,
CCH3), 123.8e133.7 (CPh). 31P NMR (162 MHz, CD2Cl2):
(s), ꢀ143.9 (sept, 1JP,F ¼ 709.0 Hz, PF6).
d
ꢀ31.9
3.5. In vitro antitumoral studies
3.5.1. Reagents and cells
3.4. X-ray crystallography
Fetal calf serum (FCS), RPMI-1640, phosphate-buffered saline
(PBS), dimethyl sulfoxide (DMSO), and propidium iodide (PI) were
obtained from Sigma (St. Louis, MO). Annexin V-FITC (AnnV) was
from Biotium (Hayward, CA). Acridin orange (AO) was from Labo-
Moderna (Paris, France). Apostat was purchased from R&D (R&D
Systems, Minneapolis, MN USA). Human thyroid carcinoma 8505C,
submandibular gland carcinoma A253, breast adenocarcinoma
MCF7, melanoma 518A2 and colon cancer SW480 were obtained
from ATCC. Cells were routinely maintained in HEPES-buffered
Data for X-ray diffraction analyses of single crystals of 2 were
collected on an Oxford Gemini S diffractometer at 293 K using Cu-
ꢁ
K
a
radiation (
l
¼ 1.54184 A, graphite monochromator). A summary
of the crystallographic data, the data collection parameters, and the
refinement parameters are given in Table 3. Multiscan corrections
were applied using the SCALE3 ABSPACK (Tmin/Tmax: 0.45/1.00)
[43,44]. The structure was solved with direct methods using
SHELXS-97 and refined using full-matrix least-square routines
against F2 with SHELXL-97 [45,46]. All non-hydrogen atoms were
refined with anisotropic displacement parameters and hydrogen
atoms with isotropic ones. H atoms were placed in calculated po-
sitions according to the riding model. The PhS(O)CH2 group was
found to be disordered over two positions with occupancies of 63%
and 37%. Furthermore, some restraints had to be used for the
refinement of the sulfinyl group, methyl group, and the phenyl ring
(DELU, SIMU, ISOR). CCDC 936713 contains the supplementary
crystallographic data for this paper. These data can be obtained free
RPMI-1640 medium supplemented with 10% FCS, 2 mM L-gluta-
mine, 0.01% sodium pyruvate, 5 ꢃ 10ꢀ5 M 2-mercaptoethanol, and
antibiotics (culture medium) at 37 ꢁC in a humidified atmosphere
with 5% CO2. After standard trypsinization, cells were seeded at
1 ꢃ104e2.5 ꢃ 103/well in 96-well plates for viability determination
and 1.5 ꢃ 105/well in 6-well plate for flow cytometry. Stock solu-
tions of investigated compounds were prepared in dimethyl sulf-
oxide (DMSO, Sigma Aldrich) at a concentration of 20 mM, filtered
through Millipore filter, 0.22
mm, before use, and diluted by
nutrient medium to various working concentrations. Final con-
centrations achieved in treated wells were 0.78, 1.56, 3.12, 6.25,
12.5, 25.0 and 50.0 mM.
Table 3
Crystallographic data, data collection parameters, and refinement parameters for 2.
3.5.2. Determination of cell viability by sulphorhodamine assay
(SRB)
2
Empirical formula
Mr
C29H32Cl2IrOPS
722.68
Orthorhombic
P212121
8.5463(7)
17.9984(13)
18.2025(14)
2799.9(4)
4
1.714
12.381
1424
3.4438e65.8701
5488
The viability of adherent viable cells was measured by SRB assay
[38]. Cells were exposed to a wide range of doses of the drugs for
96 h and then fixed with 10% of TCA for 2 h at 4 ꢁC. After fixation,
cells were washed in distilled water, stained with 0.4% SRB solution
30 min at RT, washed and dried overnight. The dye was dissolved in
10 mM TRIS Buffer and the absorbance was measured at 540 nm
with the reference wavelength at 640 nm. Results are expressed as
percentage of control that was arbitrarily set to 100%.
Crystal System
Space group
ꢁ
a/A
ꢁ
b/A
ꢁ
c/A
V/A
3
ꢁ
Z
Dcal/g cmꢀ3
m
(Cu-K
F(000)
range/ꢁ
a
)/mmꢀ1
3.5.3. Cell cycle analysis
Cells were treated with IC50 dose of [Ir(
Cl2{Ph2PCH2S(O)Ph- P}] (2) for 48 h, trypsinized, and fixed in 70%
ethanol at 4 ꢁC over-night. Cells were thoroughly washed in PBS,
h
5-C5Me5)
q
Rfln collected
Refln observed [I > 2
Rfln independent
k
s
(I)]
3568
3829 (Rint ¼ 0.0395)
3829/397/358
1.040
0.0564, 0.1437
0.0601, 0.1464
1.870/ꢀ1.191
and stained with solution containing PI (20
mg/ml) and RNase
Data/restraints/parameters
Goodness-of-fit on F2
(0.1 mg/ml) for 30 min at 37 ꢁC in dark. Red fluorescence was
analyzed with FACSCalibur flow cytometer (BD, Heildelberg, Ger-
many). The distribution of cells in different cell cycle phases was
determined with Cell Quest Pro software (BD) [47].
R1, wR2 [I > 2
R1, wR2 (all data)
Largest diff. peak and hole/e A
s(I)]
ꢁꢀ3

222
G. Ludwig et al. / European Journal of Medicinal Chemistry 69 (2013) 216e222
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h
5-C5Me5)
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m
M DHR for 20 min before treatment
with [Ir(
h
5-C5Me5)Cl2{Ph2PCH2S(O)Ph-
k
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G. L. gratefully acknowledges financial support from Gra-
duiertenförderung des Landes Sachsen-Anhalt. The authors (Uni-
versity of Belgrade) would like to acknowledge financial support
from the Ministry of Science and Technological Development of the
Republic of Serbia (Grant No. 173013) and Dr. Jürgen Schmidt
(Department of Bioorganic Chemistry, Leibniz Institute of Plant
Biochemistry) for the high-resolution ESI mass spectra.
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
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ꢀ