Anticancer potential of (pentamethylcyclopentadienyl)chloridoiridium(III) complexes bearing κp and κp,κS-coordinated Ph 2PCH2CH2CH2S(O)xPh (x=0-2) ligands

Article abstract of DOI:10.1002/cmdc.201300479

Iridium(III) complexes of the type [Ir(η⁵-C ₅Me₅)Cl₂{Ph₂PCH₂CH ₂CH₂S(O)_xPh-κP}] (x=0-2; 1-3) and [Ir(η⁵-C₅Me₅)Cl{Ph₂

Full text of DOI:10.1002/cmdc.201300479

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DOI: 10.1002/cmdc.201300479
Anticancer Potential of
(Pentamethylcyclopentadienyl)chloridoiridium(III)
Complexes Bearing kP and kP,kS-Coordinated
Ph2PCH2CH2CH2S(O)xPh (x=0–2) Ligands
[a]
[b]
[b]
[b]
´
´
´
´
Gerd Ludwig, Ivan Randelovic, Danijela Maksimovic-Ivanic, Sanja Mijatovic,
¯
[b]
[b]
[c]
[c]
[a]
´
´
Mirna Z. Bulatovic, Djordje Miljkovic, Marcus Korb, Heinrich Lang, Dirk Steinborn,
[a]
´
and Goran N. Kaluderovic*
¯
Iridium(III) complexes of the type [Ir(h5-C5Me5)Cl2{Ph2PCH2-
CH2CH2S(O)xPh-kP}] (x=0–2; 1–3) and [Ir(h5-C5Me5)Cl{Ph2PCH2-
CH2CH2S(O)xPh-kP,kS}][PF6] (x=0–1; 4 and 5) with 3-(diphenyl-
phosphino)propyl phenyl sulfide, sulfoxide, and sulfone ligands
Ph2PCH2CH2CH2S(O)xPh were designed, synthesized, and char-
acterized fully, including X-ray diffraction analyses for com-
plexes 3 and 4. In vitro studies against human thyroid carcino-
ma (8505C), submandibular carcinoma (A253), breast adenocar-
cinoma (MCF-7), colon adenocarcinoma (SW480), and melano-
ma (518A2) cell lines provided evidence for the high biological
potential of the neutral and cationic iridium(III) complexes.
Neutral iridium(III) complex 5 proved to be the most active,
with IC50 values up to about 0.1 mm, representing activities of
up to one order of magnitude higher than cisplatin. Using
8505C cells, apoptosis was shown to be the main mechanism
through which complex 5 exerts its tumoricidal action. The de-
scribed iridium(III) complexes represent potential leads in the
search for novel metal-based anticancer agents.
Introduction
Bioinorganic chemistry is a fascinating and creative area full of
possibilities.[1] One of the earliest examples, which clarifies this
statement, was the discovery of the biological potential of cis-
platin by Rosenberg in 1965.[2,3] Cisplatin itself can still be con-
sidered the “gold standard” for all metal-based anticancer
drugs that followed.[4] Despite the success of cisplatin and
other platinum-based anticancer drugs, further transition-metal
complexes were examined for their cytotoxic activity. This can
be explained, among other things, by the drawbacks of cispla-
tin, such as dose-dependent side effects and the partial resist-
ance of some carcinomas.[5–10] Thus, other metal-based anti-
cancer agents (M=Ti, Ru, Au, …) were tested for their biologi-
cal potential, and compounds like the octahedral ruthenium
complexes [imiH]trans-[Ru(imi-kN)(DMSO-kS)Cl4] (imi=imida-
zole; DMSO=dimethylsulfoxide) and [indH]trans-[Ru(ind-
kN)2Cl4] (ind=indazole) demonstrated their potential and en-
tered clinical trials.[11–17] The analogous iridium complexes,
[imiH]trans-[Ir(imi-kN)(DMSO-kS)Cl4] and [indH]trans-[Ir(ind-
kN)2Cl4],[18] and other iridium(III) complexes proved to be less
active or inactive[19–26] due to the kinetic inertness of low-spin
d6 IrIII complexes.[27] Nevertheless, iridium-based anticancer
_
_
agents, such as the fac-[Ir(NN)(DMSO-kS)Cl3] (NN=diimine-type
ligand as in I), with high biological potential have been pre-
pared.[28] Further steps were taken in the field of iridium-based
anticancer agents with the preparation of h5-pentamethylcy-
clopentadienyl iridium(III) complexes of the type [Ir(h5-
_
_
C5Me5)Cl(XY)]n+ (XY=bidentate ligand), but these complexes
_
with N,N’ ligands (XY=ethylendiamine, 2,2’-bipyridine, 1,10-
phenanthroline)[29] and [Ir(h5-C5Me5)Cl2(PTA)] (PTA=1,3,5-triaza-
7-phosphaadamantane)[24] showed no activity when evaluated
in vitro cytotoxic studies. As presented by Sheldrick et al.[30]
and Sadler et al.,[29] the incorporation of a functionalized bipyri-
dine/phenanthroline or C5Me5 ligand in the above-mentioned
types of compounds resulted in highly active iridium(III) com-
plexes (e.g., complexes II and III). Another way to increase the
´
[a] G. Ludwig, Prof. Dr. D. Steinborn, Prof. Dr. G. N. Kaluderovic
¯
cytotoxic activity of these complexes is to exchange the neu-
_
Institute of Chemistry, Martin Luther University Halle-Wittenberg
Kurt-Mothes-Straße 2, 06120 Halle (Germany)
tral diimine-type ligand for an anionic NC ligand (complex IV).
For this type of complex, IC50 values in the range exhibited by
cisplatin were observed.[31,32]
E-mail: goran.kaluderovic@chemie.uni-halle.de
´
´
´
´
´
[b] I. Randelovic, Dr. D. Maksimovic-Ivanic, Dr. S. Mijatovic, M. Z. Bulatovic,
¯
Previously, our group made some promising steps in the
field of iridium-based anticancer compounds by showing the
biological potential of neutral and cationic iridium(III) com-
plexes with (diphenylphosphino)methyl phenyl sulfide, sulfox-
ide and sulfone ligands (V and VI).[33] Here, we report the
design, synthesis and biological potential of neutral and cat-
ionic iridium(III) complexes with 3-(diphenylphosphino)propyl
´
Dr. D. Miljkovic
Institute for Biological Research “Sinisa Stankovic”, University of Belgrade
Bulevar despota Stefana 142, 11060 Belgrade (Serbia)
[c] Prof. M. Korb, Prof. Dr. H. Lang
Institute of Chemistry, Chemnitz University of Technology
Straße der Nationen 62, 09111 Chemnitz (Germany)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300479.
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The complexes were characterized by elemental analyses,
1H, 13C, and 31P NMR spectroscopy, and single-crystal X-ray
structure analyses (for 3 and 4). Selected NMR spectroscopic
data for complexes 1–5 are given in Table 1. For the neutral
iridium(III) complexes, coordination-induced shifts (CIS) of the
phosphorus resonances (dP(coord.)–dP(uncoord.)) between d=13 and
14 ppm were observed. In contrast, the CIS of the cationic
iridium(III) complexes 4 and 5 (kP,kS-coordination) were found
1
to be much smaller (ca. d=2 ppm). H and 13C chemical shifts
of the ligands are largely independent from the type of the co-
ordination (kP vs kP, kS), and the resonances of the h5-C5Me5
ligand also proved to be virtually independent of the type of
S(O)x functionalization.
Crystals of [Ir(h5-C5Me5)Cl2{Ph2PCH2CH2CH2S(O)2Ph-kP}] (3)
and [Ir(h5-C5Me5)Cl{Ph2PCH2CH2CH2SPh-kP,kS}][PF6] (4) suitable
for X-ray diffraction analyses were obtained from solutions of
dichloromethane/n-pentane at room temperature. Neutral
Table 1. Selected NMR spectroscopic data of [Ir(h5-C5Me5)Cl2{Ph2PCH2CH2CH2S(O)xPh-
kP}] (1–3) and [Ir(h5-C5Me5)Cl{Ph2PCH2CH2CH2S(O)xPh-kP,kS}][PF6] (4 and 5).
phenyl sulfide, sulfoxide and sulfone ligands of the
type [Ir(h5-C5Me5)Cl2{Ph2PCH2CH2CH2S(O)xPh-kP}] (x=
0–2; complexes 1–3) and [Ir(h5-C5Me5)Cl{Ph2PCH2-
CH2CH2S(O)xPh-kP,kS}][PF6] (x=0–1; complexes 4 and
5). Furthermore, structure–activity relationships sur-
rounding the spacer length (CH2CH2CH2 vs CH2) and
the oxidation state of the sulfur atom (SPh vs S(O)Ph
vs S(O)2Ph) are discussed.
[a]
Compd
Ph2PCgH2CbH2CaH2S(O)xPh[a]
da-C db-C dP
g-C (1JP,C
C5Me5
dCMe (3JP,C
x[b]
d
)
dH (4JP,H
)
)
dC (2JP,C
)
1
2
3
4
5
0 (kP) 37.2 21.9 25.1 (36.2) À2.4 1.26 (2.4)
1 (kP) 58.0 17.6 26.9 (33.6) À3.0 1.33 (2.2)
2 (kP) 37.2 24.2 56.2 (35.5) À2.4 1.21 (2.2)
0 (kP,kS) 37.5 22.3 25.7 (36.4) À14.5 1.22 (2.4)
1 (kP,kS) 55.5 17.2 25.1 (37.4) À14.1 1.26 (2.4)
8.2 (0.7)
8.1 (1.0)
8.7 (0.7)
8.3 (0.7)
8.0
97.3 (2.1)
92.1 (2.9)
96.3 (2.6)
97.8 (2.1)
100.7 (1.5)
[a] d values are given in ppm; the corresponding J value in Hz is given in parentheses.
[b] The coordination mode of the ligand is given in parentheses.
Results and Discussion
iridium(III) complex 3 crystallized as an isolated molecule with-
out unusual intermolecular interactions (shortest distance be-
tween non-hydrogen atoms: O1···C4’ 2.975(9) ꢁ). Cationic
iridium(III) complex 4 crystallized in discrete cations and anions
(shortest distance between non-hydrogen atoms: C2···F2
2.982(4) ꢁ). The structures of the two complexes are presented
in Figure 1, and selected structural parameters are given in the
figure caption.
Syntheses and characterization
Neutral iridium(III) complexes of the type [Ir(h5-C5Me5)Cl2-
{Ph2PCH2CH2CH2S(O)xPh-kP}] (1–3) bearing kP-coordinated li-
gands with pendant sulfide, sulfoxide and sulfone groups were
synthesized according to Scheme 1a. Under cleavage of the Ir–
Cl–Ir bridges of the starting dinuclear iridium complex, forma-
tion of the mononuclear complexes 1–3 could be afforded in
yields between 63 and 74%. The desired cationic complexes of
the type [Ir(h5-C5Me5)Cl{Ph2PCH2CH2CH2S(O)xPh-kP,kS}][PF6] (4
and 5) were accessed through the subsequent reaction of neu-
tral iridium(III) complexes 1 and 2 with [NH4][PF6] (Scheme 1b)
in yields of 63% (4) and 70% (5). All complexes were found to
be stable in air over a period of weeks and soluble in DMSO
and in dichloromethane.
The main structural feature is that both complexes adopt
a half-sandwich (“piano stool”) conformation. In the case
of neutral complex 3, the coordination sphere of iridium(III)
is built up by an h5-C5Me5 ligand (distance Ir···ring centroid:
1.823 ꢁ), two chlorido ligands, as well as the Ph2PCH2-
CH2CH2S(O)2Ph-kP ligand. The coordination sphere of iridium-
(III) in complex 4 is completed by an h5-C5Me5 ligand (distance
Ir···ring
centroid:
centroid
1.849 ꢁ), a chlorido ligand, as
well as the chelating ligand
Ph2PCH2CH2CH2SPh-kP,kS.
In
complex 3, the angles at the
iridium(III) atom are close to 908
(87.8(7)–90.4(7)8), so the struc-
ture can be considered a distort-
ed octahedron. Due to the kP,kS
coordination of the ligand (S1–
Ir–P1 87.4(3)8) in 4, the devia-
Scheme 1. Synthetic routes to iridium(III) complexes bearing Ph2PCH2CH2CH2S(O)xPh-kP (1–3) and
Ph2PCH2CH2CH2S(O)xPh-kP, kS (4 and 5) ligands.
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Figure 1. Single-crystal X-ray diffraction structures of a) [Ir(h5-C5Me5)Cl{Ph2PCH2CH2CH2S(O)2Ph-kP}] in crystals of 3 and b) [Ir(h5-C5Me5)Cl{Ph2PCH2CH2CH2SPh-
kP,kS}]+ in crystals of 4. The ellipsoids are shown with a probability of 50%. H atoms have been omitted for clarity. Selected structural parameters (distances
in ꢁ, angles in 8): Ir1–Cl1 2.424(2), Ir1–Cl2 2.418(2), Ir1–P1 2.302(2), Cl1–Ir1–Cl2 90.4(7), Cl1–Ir–P1 88.0(7), Cl2–Ir–P1 87.8(7), C25–S1–C26 105.2(4), O1–S1–O2
118.5(4) for complex 3; Ir1–Cl1 2.402(9), Ir1–S1 2.357(9), Ir1–P1 2.310(1), Cl1–Ir1–S1 93.2(3), Cl1–Ir–P1 86.5(3), S1–Ir–P1 87.4(3), C11–S1–C17 97.92(17), C11–S1–
Ir1 112.58(1), C17–S1–Ir1 97.9(2) for complex 4.
tions of 908 are slightly higher (86.5(3)–93.2(3)8). The six-mem-
bered IrPC3S iridacycle in 4 possesses a chair conformation,
with atoms Ir1 and C17 forming the apices. Bond lengths of
IrÀCl (2.402(9)–2.424(3) ꢁ) and IrÀP (2.302(3)/2.310(1) ꢁ) in both
complexes, and additionally IrÀS (2.357(9) ꢁ in complex 4, are
in the expected range (median Ir–Cl: 2.419 ꢁ, lower/upper
quartile: 2.315/2.606 ꢁ, n=543; median Ir–P: 2.316 ꢁ, lower/
upper quartile: 2.151/2.462 ꢁ, n=543; median Ir–S: 2.357 ꢁ,
lower/upper quartile: 2.244/2.423 ꢁ, n=148).[34]
sulfone L3 ligands possess 10- to 400-times lower activities in
vitro than the related iridium(III) complexes 1–5 (cf. data in
Table 1 vs Table S1 in the Supporting Information).[36] Except
for complex 3, both the neutral and cationic iridium complexes
exhibited an up to 25-times higher antiproliferative activity
than cisplatin; even against cisplatin-resistant cell lines 8505C,
MCF-7 and SW480 high activities were found. In the examined
series, the iridium(III) complex with the highest activity proved
to be complex 5 against the MCF-7 cell line, with an IC50 value
of 0.1 mm (c.f., cisplatin: IC50 =2.0 mm). For the neutral iridium
complexes, a direct correlation between the oxidation state of
the pendant S(O)xPh (x=0–2) group of the ligand and the cy-
totoxic activity could be determined. Namely, the in vitro activ-
ity increases in the following order: S(O)2 <S(O)<S. This trend
is particularly apparent when comparing the IC50 values of
iridum(III) complexes 1–3 against the 8505C cell line. Thus,
neutral iridium(III) complex 1 (n/x=3/0) with a kP coordinated
ligand and a sulfide group is 30-times more active than com-
plex 3 (n/x=3/2), with a pendant sulfonyl group.
Biological studies
To determine the biological potential of iridium(III) complexes
1–5, in vitro cytotoxicity studies were performed against
human thyroid carcinoma (8505C), submandibular carcinoma
(A253), breast adenocarcinoma (MCF-7), colon adenocarcinoma
(SW480), and melanoma (518A2) cell lines. The cells were cul-
tured in the presence of various concentrations of the corre-
sponding iridium complexes, and the cell viability was ana-
lyzed using sulforhodamine B (SRB) microculture col-
orimetric assay.[35] According to the obtained results,
Table 2. Cytotoxicity of iridium(III) complexes 1–5 in comparison with cisplatin
all complexes cause a dose-dependent decrease in
cell viability (Figure 2). The IC50 values of iridium(III)
complexes 1–5 are listed in Table 2; the activities of
cisplatin are included for comparison. For further un-
derstanding, the IC50 values of the corresponding li-
gands L1–L3 and related ruthenium(II) complexes are
given in Table S1 in the Supporting Information.[36,37]
Furthermore, IC50 values of analogous iridium(III)
complexes bearing a methylene spacer (Ph2PCH2-
S(O)xPh), instead of Ph2PCH2CH2CH2S(O)xPh ligands
L1–L3, are presented in Table S2 in the Supporting
Information.[33]
against a panel of cancer cell lines.[a]
Compd
x[b]
IC50 [mm][c]
518A2
8505C
A253
MCF-7
SW480
1
2
3
4
0 (kP)
1 (kP)
2 (kP)
0.3Æ0.1
0.7Æ0.4
4.4Æ2.5
1.0Æ0.6
0.4Æ0.2
1.5Æ0.2
0.2Æ0.0
0.6Æ0.0
5.8Æ1.3
0.5Æ0.2
0.3Æ0.1
5.0Æ0.2
0.2Æ0.0
0.5Æ0.1
4.9Æ0.1
0.4Æ0.0
0.3Æ0.1
0.8Æ0.1
0.2Æ0.1
0.3Æ0.0
6.7Æ1.4
0.4Æ0.1
0.1Æ0.1
2.0Æ0.1
0.6Æ0.2
1.0Æ0.4
4.5Æ0.1
1.0Æ0.3
0.9Æ0.4
3.2Æ0.2
0 (kP, kS)
1 (kP, kS)
5
Cisplatin
[a] Human thyroid carcinoma 8505C, submandibular gland carcinoma A253, breast ad-
enocarcinoma MCF7, melanoma 518A2 and colon cancer SW480. [b] The coordination
mode of the ligand is given in parentheses. [c] The compound concentration required
to inhibit 50% cell growth (IC50). Data represent the mean Æ standard deviation (SD)
of three independent experiments performed in triplicate.
Without exception, non-coordinated phosphino-
sulfide L1, phosphino-sulfoxide L2 and phosphino-
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Figure 2. Representative graphs showing survival (%) of cells grown for 96 h in the presence of increasing concentrations of compounds 1–5.
A direct relationship was also observed between the length
of the alkanediyl spacer and cytotoxic activity. Thus, for both
the neutral and the cationic iridium(III) complexes, the com-
plexes with a trimethylene spacer possess an up to nearly two
orders of magnitude higher activity than the complexes with
a methylene spacer (cf. Table 1 vs Table S2 in the Supporting
Information).[33] Neutral iridium(III) complexes 1–3 exhibited
higher activities (up to two orders of magnitude) against all
tested cell lines than the related ruthenium(II) complexes,
[Ru(h6-p-cym)Cl2{Ph2PCH2CH2CH2S(O)xPh-kP}] (x=0, 1, 2) (cf.
Table 1 vs Table S1 in the Supporting Information).[36] In con-
trast, the same tendency was not observed for cationic iridium-
(III) complexes 4 and 5. These complexes exhibited only a simi-
lar or slightly better cytotoxic activity in comparison with the
corresponding cationic ruthenium complexes of the type
[Ru(h6-p-cym)Cl{Ph2PCH2CH2CH2SPh-kP,kS}][PF6] (x=0, 1).[37]
For further analysis, a thyroid carcinoma (8505C) cell line
was selected as a model of a highly malignant and almost in-
curable type of tumor. 8505C cells were treated with the IC50
dose of complex 5 for 48 hours in order to explore the mecha-
nism of action of iridium(III) complexes. Induction of apoptosis
was analyzed with double Annexin V/propidium iodide (PI)
staining. In comparison to the untreated control cells (early
apoptosis 1.4%, late apoptosis/necrotic cells 5.7%), treatment
with 5 for 48 hours gave rise to an increase in both early apop-
totic cells (7.8%) marked by externalization of phosphatidylser-
in, stained by Annexin V, and late apoptotic/necrotic cells
(36.5%), stained by both Annexin V and PI (Figure 3a). 8505C
cells treated with complex 5 exhibited increased activation of
caspases (5: 58.3%; control: 7.7%), indicating that caspase-de-
pendent apoptosis is probably behind the action of this com-
pound (Figure 3b). From the cell-cycle analysis, an increased
percentage of hipodiploid cells with fragmented DNA upon
treatment with 5 was observed (Figure 3c). The mechanism of
cell death induced by complex 5 seems to be similar to the
previously evaluated iridium(III) complex [Ir(h5-C5Me5)Cl2-
{Ph2PCH2S(O)Ph-kP}].[33] Thus, these results confirm that apop-
tosis is the dominant mode of action of this kind of iridium-
based cytotoxic agents. While these compounds induce cell
death through the same pathway, some differences in their
biological effects could be observed. Complex 5 inhibits cell vi-
ability by 50% at a much lower concentration than [Ir(h5-
C5Me5)Cl2{Ph2PCH2S(O)Ph-kP}], and it also induces significant
apoptotic tumor cell death. In the cytoplasm of 8505C cells ex-
posed to compound 5, only a moderate enhancement of au-
thophagic process was detected (5: 2.3%; control: 0.4%; Fig-
ure 3d). In contrast to [Ir(h5-C5Me5)Cl2{Ph2PCH2S(O)Ph-kP}], au-
tophagy process is irrelevant for the effectiveness of complex
5.
Previously, it was shown that [Ir(h5-C5Me5)Cl2{Ph2PCH2S(O)Ph-
kP}] complex significantly decreased levels of free radicals—re-
active oxygen species (ROS) and reactive nitrogen species
(RNS)—in 8505C cells (24 h: 90%; 48 h: 95%).[33] In order to ex-
plore the involvement of oxidative stress in the cytotoxicity of
5, cells were stained with the specific dye dihydrorhoda-
mine 123 (DHR), which is sensitive to ROS and RNS. The cumu-
lative effect was measured after 24 and 48 hours of exposure
to complex 5. The results obtained were in accordance with
previously observed decreases in ROS/RNS levels in 8505C cells
upon treatment with [Ir(h5-C5Me5)Cl2{Ph2PCH2S(O)Ph-kP}]. Com-
plex 5 decreased free radical levels in cells by more than 40%
in comparison with control cells (Figure 4). From the literature,
it is known that thyroid cancer cells treated with bortezomib,
a
proteasome inhibitor, demonstrated this phenomenon,
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which was explained as a cell-specific response to toxic stimu-
li.[38,39] Namely, a decrease in the ROS level of thyroid cancer
cells exposed to cytotoxic stimuli
sulfur groups in vivo, the active species are unknown and
the order of activities could vary in vivo.
might occur as a result of elevat-
ed redox protection. Neverthe-
less, as in the case of [Ir(h5-
C5Me5)Cl2{Ph2PCH2S(O)Ph-kP}]
complex, this protection of the
cells against oxidative stress did
not have an influence on the ef-
fectiveness of complex 5.
Conclusions
In the current study, neutral and
cationic iridium(III) complexes of
the type [Ir(h5-C5Me5)Cl2{Ph2-
PCH2CH2CH2S(O)xPh-kP}] (x=0–
2; 1–3) and [Ir(h5-C5Me5)Cl-
{Ph2PCH2CH2CH2S(O)xPh-kP,kS}]-
[PF6] (x=0–1; 4 and 5) with 3-
(diphenylphosphino)propyl
phenyl sulfide, sulfoxide and sul-
fone ligands were synthesized,
and their biological potential
was proven against several cell
lines. The following conclusions
can be drawn (for an overview,
see Figure 5):
1. The cytotoxic activities of ex-
amined iridium(III) complexes
1, 2, 4, and 5 were found to
be up to 25 times higher
than that of cisplatin and up
to 400 times higher than
those of the corresponding li-
gands (L1–L3).
2. The different coordination
mode (kP vs. kP,kS) of the
Ph2PCH2CH2CH2S(O)xPh ligand
(x=0, 1) does not significant-
ly affect the cytotoxic activity
of the iridium(III) complexes.
3. The oxidation state of sulfur
in the ligands (S(O)2 <S(O)<
S) and the length of the
spacer between the P and
S atoms (CH2 <(CH2)3) have
a
greater impact on the
cytotoxic activities of the
iridium(III) complexes in that
the complexes with the Ph2-
PCH2CH2CH2SPh and PPh2-
CH2S(O)Ph ligands are the
most active. Due to a possible
Figure 3. The effect of [Ir(h5-C5Me5)Cl{Ph2PCH2CH2CH2S(O)Ph-kP,kS}][PF6] (5) on malignant cell death. 8505C cells
were exposed to IC50 doses of compound 5, and a) apoptosis, b) caspase activation, c) cell cycle distribution,
oxidation of the pendant d) presence of autophagic vesicles were investigated.
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dried (n-pentane over Na/benzophenone; MeOH over magnesium;
CH2Cl2 over CaH2) and freshly distilled prior to use. NMR spectra
(1H, 13C, 31P) were recorded at 278C on Varian Gemini 200 and VXR
400 spectrometers. Chemical shifts (d) are reported in parts per
million (ppm) relative to residual solvent signals (CD2Cl2: dH =
5.32 ppm, dC =53.8 ppm); dP values are relative to H3PO4 (85%) as
an external reference. Microanalyses (C, H) were performed in the
Microanalytical Laboratory of the Martin Luther University Halle-
Wittenberg (Germany) using a CHNS-932 (LECO) elemental ana-
lyzer. [{IrCl2(h5-C5Me5)}2] and the ligands were prepared according
to literature procedures.[40,41]
Preparation of [IrCl2(h5-C5Me5){Ph2PCH2CH2CH2S(O)xPh-kP}] (1–3):
A solution of [{IrCl2(h5-C5Me5)}2] (127 mg, 0.16 mmol) in CH2Cl2
(25 mL) was treated while stirring with the appropriate ligand
(0.32 mmol), and the reaction was stirred at RT overnight. The re-
sultant orange precipitate was isolated by filtration, washed with
n-pentane (3ꢂ2 mL), and dried under vacuum to give the desired
complexes as reddish powders.
Figure 4. The effect of [Ir(h5-C5Me5)Cl{Ph2PCH2CH2CH2S(O)Ph-kP,kS}][PF6] (5)
on the levels of reactive oxygen (ROS) and nitrogen (RNS) species in 8505C
cells: control (&) and complex 5 (&).
1
4. In all cell lines tested, neutral iridium(III) complexes 1–3
possess higher cytotoxic activities in comparison with the
corresponding ruthenium(II) complexes [Ru(h6-p-cym)-
Cl2{Ph2PCH2CH2CH2S(O)xPh-kP}] (x=0, 1, 2).
Complex 1 (x=0): Yield: 153 mg (65%); H NMR (400 MHz, CD2Cl2):
4
d=1.26 (d, JP,H =2.43 Hz, 15H, CH3), 1.29–1.34 (m, 1H, CH2CH2CH2),
2.53–2.62 (m, 2H, CH2CH2CH2 +CH2PPh2), 3.07–3.12 (m, 1H, SCH2),
3.55–3.57 (m, 1H, CH2PPh2), 4.18–4.24 (m, 1H, SCH2), 7.51–
8.13 ppm (m, 15H, HPh); 13C NMR (100 MHz, CD2Cl2): d=8.2 (d,
3JP,C =0.7 Hz, CCH3), 21.9 (s, CH2CH2CH2), 25.1 (d, 1JP,C =36.2 Hz,
CH2PPh2), 37.2 (s, SCH2), 97.3 (d, 2JP,C =2.1 Hz, CCH3), 123.7–
135.5 ppm (CPh); 31P NMR (162 MHz, CD2Cl2): d=À2.4 ppm (s); Anal.
calcd for C31H36Cl2IrPS (MW=734.78): C, 50.67; H, 4.94, found: C,
50.27; H, 4.76.
In summary, for neutral and the cationic iridium(III) com-
plexes 1–5, a high biological potential, especially against
highly aggressive, cisplatin-resistant tumor cell lines 8505C,
MCF-7 and SW480, was observed. Importantly, apoptosis was
shown to be the main pathway through which these com-
plexes exert their cytotoxic action.
1
Complex 2 (x=1): Yield: 178 mg (74%); H NMR (400 MHz, CD2Cl2):
4
d=1.33 (d, JP,H =2.16 Hz, 15H, CH3), 1.64–1.82 (m, 2H, CH2CH2CH2),
2.59–2.78 (m, 2H, SOCH2), 2.86–3.03 (m, 2H, CH2PPh2), 7.40–7.86
(m, 11H, HPh), 7.40–7.86 ppm (m, 4H, HPh); 13C NMR (100 MHz,
3
Experimental Section
CD2Cl2): d=8.1 (d, JP,C =1.0 Hz, CCH3), 17.6 (s, CH2CH2CH2), 26.0 (d,
2
1JP,C =33.6 Hz, CH2PPh2), 58.0 (s, SOCH2), 92.1 (d, JP,C =2.9 Hz, CCH3),
Synthesis
123.9–135.5 ppm (CPh); 31P NMR (162 MHz, CD2Cl2): d=À3.0 ppm
(s); Anal. calcd for C31H36Cl2IrOPS (MW=750.78): C, 49.59; H, 4.83,
found: C, 49.78; H, 5.01.
General comments: All reactions and manipulations were carried
out under argon using standard Schlenk techniques. Solvents were
Figure 5. Range of cytotoxic activities, based on the IC50 values determined against the five cell lines under investigation, of iridium(III) complexes 1–5
([Ir]=Ir(h5-C5Me5)Clx; x=1, 2) and of the related ruthenium(II) complexes [Ru(h6-p-cym)Cl2{Ph2PCH2CH2CH2S(O)xPh-kP}] and [Ru(h6-p-cym)-
Cl{Ph2PCH2CH2CH2S(O)xPh-kP,kS}][PF6] ([Ru]=Ru(h6-p-cym)Clx; x=1, 2). For comparison, the corresponding values of cisplatin and the uncoordinated ligands
Ph2PCH2CH2CH2S(O)xPh L1–L3 are given. [a] Four values; exception: cell line MCF-7 (IC50 =1.8 mm); [b] Four values; exception: cell line MCF-7 (IC50 =23.0 mm).
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1
Complex 3 (x=2): Yield: 174 mg (71%); H NMR (400 MHz, CD2Cl2):
In vitro assays
4
d=1.21 (d, JP,H =2.19 Hz, 15H, CH3), 1.52–1.62 (m, 2H, CH2CH2CH2),
2.74–2.80 (m, 2H, SO2CH2), 2.84–2.88 (m, 2H, CH2PPh2), 7.37–
7.71 ppm (m, 15H, HPh); 13C NMR (100 MHz, CD2Cl2): d=8.7 (d,
3JP,C =0.7 Hz, CCH3), 24.2 (s, CH2CH2CH2), 37.2 (s, SO2CH2), 56.2 (d,
1JP,C =35.5 Hz, CH2PPh2), 96.3 (d, 2JP,C =2.6 Hz, CCH3), 126.7–
133.7 ppm (CPh); 31P NMR (162 MHz, CD2Cl2): d=À2.4 ppm (s); Anal.
calcd for C31H36Cl2IrO2PS (MW=766.78): C, 48.56; H, 4.73, found: C,
48.21; H, 4.41.
Reagents and cells: Fetal calf serum (FCS), RPMI-1640, phosphate-
buffered saline (PBS), dimethyl sulfoxide (DMSO), and propidium
iodide (PI) were obtained from Sigma (St. Louis, MO, USA). Annexin
V-FITC (AnnV) was purchased from Biotium (Hayward, CA, USA). Ac-
ridin orange (AO) was obtained from Labo-Moderna (Paris, France).
Apostat was purchased from 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 the ATCC. Cells are routinely
maintained in 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic
acid (HEPES)-buffered RPMI-1640 medium supplemented with 10%
FCS, 2 mm l-glutamine, 0.01% sodium pyruvate, and antibiotics
(culture medium) at 378C in a humidified atmosphere with 5%
CO2. After standard trypsinization, cells were seeded at 1ꢂ103–
2.5ꢂ103/well in 96-well plates for viability determination and 1.5ꢂ
105/well in six-well plate for flow cytometry.
Preparation of [IrCl(h5-C5Me5){Ph2PCH2CH2CH2S(O)xPh-kP,kS}][PF6]
(4 and 5): A solution of [{IrCl2(h5-C5Me5)}2] (127 mg, 0.16 mmol) in
MeOH (30 mL) was treated while stirring with the appropriate
ligand (0.32 mmol), and the reaction was heated at refux for 3 h.
Next, [NH4][PF6] (6 equiv) was added, and the reaction mixture was
stored at À708C overnight. The resultant precipitate was isolated
by filtration, washed with Et2O (3ꢂ2 mL), and dried under vacuum
to give the desired complexes as yellow powders.
1
Complex 4 (x=0): Yield: 170 mg (63%); H NMR (400 MHz, CD2Cl2):
4
d=1.22 (d, JP,H =2.39 Hz, 15H, CH3), 1.30–1.38 (m, 1H, CH2CH2CH2),
Determination of cell viability by sulforhodamine B assay (SRB): The
viability of adherent viable cells was measured by an SRB assay.[35]
Cells were exposed to a wide range of doses of test compound for
96 h and then fixed with 10% trichloroacetic acid (TCA) for 2 h at
48C. After fixation, cells were washed with distilled water, stained
with 0.4% SRB solution for 30 min at RT, then washed and dried
overnight. The dye was dissolved in 10 mm TRIS buffer, and the ab-
sorbance 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%.
2.41–2.60 (m, 2H, CH2CH2CH2 +CH2PPh2), 2.99–3.03 (m, 1H, SCH2),
3.51–3.60 (m, 1H, CH2PPh2), 4.20–4.26 (m, 1H, SCH2), 7.52–
8.09 ppm (m, 15H, HPh); 13C NMR (100 MHz, CD2Cl2): d=8.3 (d,
3JP,C =0.7 Hz, CCH3), 22.3 (s, CH2CH2CH2), 25.7 (d, 1JP,C =36.4 Hz,
CH2PPh2), 37.5 (s, SCH2), 97.8 (d, 2JP,C =2.1 Hz, CCH3), 123.7–
135.8 ppm (CPh); 31P NMR (162 MHz, CD2Cl2): d=À14.5 (s),
À144.6 ppm (sept, 1JP,F =710.4 Hz, PF6); Anal. calcd for
C31H36ClF6IrP2S (MW=844.29): C, 44.10; H, 4.30, found: C, 43.88; H,
4.13.
1
Complex 5 (x=1): Yield: 193 mg (70%); H NMR (400 MHz, CD2Cl2):
d=1.26 (d, JP,H =2.37 Hz, 15H, CH3), 2.00–2.14 (m, 1H, CH2CH2CH2),
Cell cycle analysis: Cells were treated with an IC50 dose of [Ir(h5-
C5Me5)Cl{Ph2PCH2CH2CH2S(O)Ph-kP,kS}][PF6] (5) for 48 h, trypsinized,
and fixed in 70% ethanol at 48C overnight. Cells were thoroughly
washed in PBS, and stained with solution containing PI
(20 mgmLÀ1) and RNase (0.1 mgmLÀ1) for 30 min at 378C in the
dark. Red fluorescence was analyzed with a FACS Calibur flow cy-
tometer (BD, Heidelberg, Germany). The distribution of cells in dif-
ferent cell-cycle phases was determined with Cell Quest Pro soft-
ware (BD).[47]
4
2.43–2.66 (m, 2H, CH2CH2CH2 +CH2PPh2), 3.28–3.32 (m, 1H, SOCH2),
3.41–3.50 (m, 1H, CH2PPh2), 4.27–4.34 (m, 1H, SOCH2), 7.54–
8.14 ppm (m, 15H, HPh); 13C NMR (100 MHz, CD2Cl2): d=8.0 (s,
1
CCH3), 17.2 (s, CH2CH2CH2), 25.1 (d, JP,C =37.4 Hz, CH2PPh2), 55.5 (s,
SOCH2), 100.7 (d, 2JP,C =1.5 Hz, CCH3), 123.7–135.6 ppm (CPh);
1
31P NMR (162 MHz, CD2Cl2): d=À14.1 (s), À146.6 ppm (sept, JP,F
=
710.8 Hz, PF6); Anal. calcd for C31H36ClF6IrOP2S (MW=860.29): C,
43.28; H, 4.22, found: C, 43.51; H, 4.42.
AnnexinV-FITC/PI, AO staining and caspase detection: Cells were ex-
posed to IC50 dose of [Ir(h5-C5Me5)Cl{Ph2PCH2CH2CH2S(O)Ph-kP,kS}]-
[PF6] (5) for 48 h. After trypsinization, cells were stained with AnnV-
FITC/PI (Biotium, Hayward, CA, USA) or Apostat according to the
manufacturer’s instruction. Alternatively, cells were stained with
a solution of AO (100 mgmLÀ1) for 15 min at 378C. Cells were ana-
lyzed with a FACS Calibur flow cytometer using Cell Quest Pro soft-
ware.
X-ray crystallography
Data for X-ray diffraction analyses of single crystals of complexes 3
and 4 were collected on an Oxford Gemini S diffractometer at
100 K using MoKa radiation (l=0.71073 ꢁ, graphite monochroma-
tor). A summary of the crystallographic data, the data collection
parameters, and the refinement parameters are given in Table S3
of the Supporting Information. Multiscan absorption corrections
were applied using SCALE3 ABSPACK (3: Tmin/Tmax =0.69/1.00; 4:
0.55/1.00).[43,44] The structures were 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 re-
fined with anisotropic displacement parameters, and hydrogen
atoms with isotropic ones. Hydrogen atoms were placed in calcu-
lated positions according to the riding model. In complex 4, the
hydrogen atoms of the methyl groups were found to be disor-
dered over two positions with occupancies of 50%. Furthermore,
some restraints had to be used for the refinement of the Ir(h5-
C5Me5) fragment (DELU, SIMU, ISOR) in complex 4. CCDC 972711
and 972712 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.
Measurement of ROS generation: The production of reactive oxygen
and nitrogen species was determined by redox-sensitive dye, dihy-
drorhodamine 123 (DHR). The cells were stained with 1 mm DHR
for 20 min before treatment with [Ir(h5-C5Me5)Cl{Ph2PCH2-
CH2CH2S(O)Ph-kP,kS}][PF6] (5). After 24 or 48 h incubation, cells
were detached, washed with PBS, and the fluorescence intensity
was analyzed with a FACS Calibur flow cytometer using Cell Quest
Pro software.
Statistical analysis: Results are the mean Æ standard deviation (SD)
of triplicate observations from three experiments, unless indicated
otherwise. The statistical significance between treatments and con-
trol was analyzed by ANOVA followed by a Student-Newman-Keul’s
test; P <0.05 was considered significant.
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Keywords: apoptosis
complexes metal-based drugs
phosphorus ligands
·
cytotoxic agents
·
iridium(III)
·
·
sulfur-functionalized
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Received: November 24, 2013
Published online on January 27, 2014
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ChemMedChem 2014, 9, 1586 – 1593 1593