Ruthenium- and osmium-arene complexes of 8-substituted indolo[3,2-c] quinolines: Synthesis, X-ray diffraction structures, spectroscopic properties, and antiproliferative activity

Article abstract of DOI:10.1016/j.ica.2012.06.004

Six novel ruthenium(II)- and osmium(II)-arene complexes with indoloquinoline modified ligands containing methyl and halo substituents in position 8 of the molecule backbone have been synthesised and comprehensively characterised by spectroscopic methods (¹H, ¹³C NMR, UV-Vis), ESI mass spectrometry and X-ray crystallography. Binding of indoloquinolines to a metal-arene scaffold makes the products soluble enough in biological media to allow for assaying their antiproliferative activity. The complexes were tested in three human cancer cell lines, namely A549 (non-small cell lung cancer), SW480 (colon carcinoma) and CH1 (ovarian carcinoma), yielding IC₅₀ values in the 10^-6-10^-7 M concentration range after continuous exposure for 96 h. Compounds with halo substituents in position 8 are more effective cytotoxic agents in vitro than the previously reported species halogenated in position 2 of the indoloquinoline backbone. High antiproliferative activity of both series of substances may be due at least in part to their potential to act as DNA intercalators.

Full text of DOI:10.1016/j.ica.2012.06.004

Inorganica Chimica Acta 393 (2012) 252–260
Contents lists available at SciVerse ScienceDirect
Inorganica Chimica Acta
journal homepage: www.elsevier.com/locate/ica
Ruthenium- and osmium-arene complexes of 8-substituted
indolo[3,2-c]quinolines: Synthesis, X-ray diffraction structures, spectroscopic
properties, and antiproliferative activity
⇑
Lukas K. Filak, Simone Göschl, Stefanie Hackl, Michael A. Jakupec, Vladimir B. Arion
Institute of Inorganic Chemistry, University of Vienna, Währinger Strasse 42, 1090 Vienna, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Available online 15 June 2012
Six novel ruthenium(II)- and osmium(II)-arene complexes with indoloquinoline modified ligands con-
taining methyl and halo substituents in position 8 of the molecule backbone have been synthesised
1
13
and comprehensively characterised by spectroscopic methods ( H, C NMR, UV–Vis), ESI mass spectro-
metry and X-ray crystallography. Binding of indoloquinolines to a metal-arene scaffold makes the
products soluble enough in biological media to allow for assaying their antiproliferative activity. The
complexes were tested in three human cancer cell lines, namely A549 (non-small cell lung cancer),
Metals in Medicine Special Issue
Keywords:
Indolo[3,2-c]quinoline
Ruthenium(II)
Osmium(II)
Anticancer compound
À6
À7
SW480 (colon carcinoma) and CH1 (ovarian carcinoma), yielding IC50 values in the 10 –10 M concen-
tration range after continuous exposure for 96 h. Compounds with halo substituents in position 8 are
more effective cytotoxic agents in vitro than the previously reported species halogenated in position 2
of the indoloquinoline backbone. High antiproliferative activity of both series of substances may be
due at least in part to their potential to act as DNA intercalators.
Ó 2012 Elsevier B.V. All rights reserved.
1
. Introduction
The fight against cancer has made considerable progress by the
in hormone-positive breast cancer therapy [15]. In ferrocifen, one
of the phenyl rings is replaced by a ferrocenyl unit, combining
the hormone-antagonistic ligand with a metal–organic redox ac-
tive moiety. Similar attempts combining the benefits of organome-
tallic core with biologically active ligands were undertaken with
indolobenzazepines, also referred to as paullones. The paullones
were originally predicted to possess cyclin dependent kinase
(CDK)-inhibitory properties by a COMPARE analysis [16]. CDKs to-
gether with their corresponding cyclins act as cell cycle triggers,
controlling cell division [17]. By interference with this highly
balanced regulatory system, cell proliferation can be controlled.
In vitro models confirmed the CDK-inhibitory properties of the
paullones [18], and up to date a broad range of paullone derivatives
has been evaluated for biological activity [19,20]. For some paull-
ones, other intracellular targets such as glycogen synthase kinase
3b (GSK3b) and mitochondrial malate dehydrogenase (mMDH)
could be identified [21].
Indoloquinolines also attracted interest during the last few
years [22–26] due to the development of convenient preparation
routes [27]. In contrast to paullones with a folded seven-mem-
bered azepine ring, indoloquinolines are flat heteroaromatic ring
systems, in which the paullone azepine ring was replaced by a
six-membered pyridine ring. We anticipated that this transforma-
tion will alter significantly the physico-chemical and biological
properties compared to the reference (paullone) compounds.
In order to overcome their limited solubility in biocompatible
media, paullones were complexed to metal ions. Ga(III) [28], Ru(II)
introduction of targeted therapies in recent years. This treatment
modality takes advantage of certain features of malignant tumours
to selectively inhibit their growth, ideally associated with low side
effects for patients. The numerous concepts that are currently
being explored to achieve tumour targeting in bioinorganic medic-
inal chemistry include ‘activation by reduction’ in hypoxic media,
as well as ‘activation by ring opening’ in the solid tumour environ-
ment with lowered pH value [1–4]. Activation by reduction is be-
lieved to be the critical step in converting a prodrug into its
active form [5]. Well-known examples supporting this hypothesis
IV
are satraplatin, a Pt compound that reached a clinical phase III
study [6], NAMI-A [7], as well as KP1019 [8], the first ruthe-
nium(III) coordination compounds in clinical studies. Another
way to gain selectivity for malignant cells over healthy tissue is
targeting enzymes or receptors that are overexpressed in certain
tumour types, e.g. thioredoxin reductase [9], ribonucleotide reduc-
tase [10,11], DNA topoisomerase [12] or glutathione S-transferase
[
13]. Another example are ferrocifen derivatives [14], which are
based on hydroxytamoxifen, an oestrogen receptor antagonist used
⇑
Corresponding author. Tel.: +43 1 4277 52615; fax: +43 1 4277 52630.
E-mail addresses: lukas.filak@univie.ac.at (L.K. Filak), simone.goeschl@univie.ac.
at (S. Göschl), stefanie-hackl@hotmail.com (S. Hackl), michael.jakupec@univie.ac.
at (M.A. Jakupec), vladimir.arion@univie.ac.at (V.B. Arion).
0
020-1693/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ica.2012.06.004

L.K. Filak et al. / Inorganica Chimica Acta 393 (2012) 252–260
253
[
29] and Cu(II) [30] coordination compounds, as well as a series of
Ru(II)- and Os(II)-arene complexes of modified paullone ligands
31–33] are well-documented in the literature. Interestingly, CDK
Matthey, KBr from Merck, while 2-acetylpyridine, hydrazine dihy-
drochloride, hydrazine hydrate, phosphorus oxychloride, isatin,
glacial acetic acid, borane in THF, 2-aminobenzonitrile, 2-amino-
5-methylbenzonitrile, sodium perborate tetrahydrate were from
Sigma–Aldrich. All these chemicals were used as received.
[
inhibition by metal-based paullones does not necessarily parallel
their in vitro antiproliferative activity, making other intracellular
targets likely to be involved in their mechanism of action [34].
Novel SAR studies showed that some ruthenium- and osmium-
arene complexes of indoloquinolines are by a factor of 10 more ac-
tive than corresponding paullone complexes in human cancer cell
lines. It is worth noting, however, that the indoloquinoline-based
complexes with a bidentate ethylenediamine binding site are less
stable than their paullone counterparts, dissociating in aqueous
media with release of the ligand [34]. Remarkably, other ethylene-
diamine based ruthenium-arene complexes do not show propen-
sity for dissociation under similar conditions [35–37]. To increase
the thermodynamic stability and kinetic inertness of the
2.2. Synthesis
1À3
The ligands HL
were synthesized by following the literature
protocols [38,39]. Ruthenium- and osmium-arene starting com-
II
II
pounds [M(p-cymene)(Cl)(
prepared as described previously [40,41]. For preparation of
Os(p-cymene)(Cl)( -Cl)] OsO was reduced first to H [OsCl ] by
2
l-Cl)] , where M = Ru and Os , were
[
l
2
4
2
6
N
2
H
4
Á2HCl in conc. HCl [42], and then reacted with
a
-terpinene.
to the
in a Schlenk
1À3
General procedure
A
for the complexation of HL
1À3
metal-arene scaffold: The corresponding ligand HL
2
complexes, sp -hybridised N-donor atoms were introduced by
tube was flushed with argon and suspended in dry ethanol. The
corresponding metal-arene dimer was dissolved in chloroform
and added to the ethanolic ligand suspension. The reaction mixture
was stirred at room temperature under argon atmosphere (in the
case of the Os complexes, light protection was also applied). The
reaction mixture was filtered through a GF3 filter paper, and
slowly added to diethyl ether previously dried over sodium sulfate.
The precipitate formed was separated by filtration and dried
in vacuo at 50 °C.
condensation of an indoloquinoline azine with 2-formyl- or 2-ace-
tylpyridine [38]. This modification led to complexes with increased
stability in biocompatible media, while retaining the in vitro anti-
proliferative activity. Further studies on modified indoloquinolines
containing different substituents in position 2 of the molecular
backbone showed that electron-withdrawing substituents are
unfavourable for cytotoxicity, whereas an electron-donating
methyl group has no influence on antiproliferative activity. The
effect of substituents in position 8 of the indoloquinoline backbone
was studied on copper(II) complexes which were found highly cyto-
toxic with IC50 values in the nanomolar concentration range [39].
Synthesis of those ligands is depicted in Scheme 1.
1
2.2.1. [Ru(p-cymene)(HL )Cl]Cl, 1a
General procedure A: N-(8-Methyl-5,11-dihydroindolo [3,2-c]—
1
Herein we report on the synthesis of six novel ruthenium- and os-
mium-arene complexes with indoloquinoline-based ligands (1a,b–
quinolin-6-ylidene)-N’-(1-pyridin-2-yl-ethylidene)azine
(HL ,
6
120 mg, 0.33 mmol), bis((
g
-p-cymene)(chlorido)(
l
-chlorido)
3
a,b) containing substituents with different electronic properties
ruthenium(II)) (101 mg, 0.16 mmol), EtOH abs. (4 mL), CHCl
(0.3 mL), diethyl ether dried over Na SO (100 mL), stirring for
22.5 h. To remove traces of the unreacted ruthenium dimer the red
precipitate was dissolved in a minimal amount of EtOH and filtered
3
in position 8 of the indoloquinoline backbone (Scheme 2). Their anti-
proliferative activity in three human cancer cell lines, namely A549
2
4
(
(
non-small cell lung cancer), SW480 (colon carcinoma) and CH1
ovarian carcinoma) has been studied and compared to that of
through a GF3 filter paper. After addition of CHCl
was added dropwise to diethyl ether previously dried over Na
100 mL). The resulting precipitate was filtered off and dried in va-
cuo at 50 °C. Yield 154 mg, 69%. Anal. Calc. for C33 RuÁ1.5H
(M 698.65): C, 56.73; H, 5.19; N, 10.02. Found: C, 56.64; H, 5.01; N,
.94%. ESI-MS (methanol), positive: m/z 636 [MÀCl] .
3
(1 mL), the filtrate
chemically related complexes (4a,b–6a,b and others).
2
SO
4
(
H33Cl
2
N
5
2
O
2
. Experimental
r
+
9
1
11
2.1. Chemicals
6
H NMR (500 MHz, DMSO-d ): 12.99 (s, 1H, H ), 10.36 (s, 1H,
5
3
17
3
1
H ), 9.60 (d, 1H, J = 6 Hz, H ), 8.36 (d, 1H, J = 8 Hz, H ), 8.31–
8.26 (m, 1H, H ), 8.23–8.19 (m, 2H, H + H ), 7.83–7.79 (m, 1H,
H ), 7.63 (d, 1H, J = 8.3 Hz, H ), 7.61–7.57 (m, 2H, H + H ),
7.42 (ddd, 1H, J = 8 Hz, J = 6 Hz, J = 2 Hz, H ), 7.31 (dd, 1H,
1
9
7
20
Ethanol and THF were dried using standard procedures.
a
-Ter-
1
8
3
10
3
4
pinene, 2-amino-5-chlorobenzonitrile were purchased from Acros
Fisher, ruthenium trichloride and osmium tetroxide from Johnson
3
3
4
2
Scheme 1. Synthesis of the indoloquinoline modified ligands [38,39]. Reagents and conditions: (i) BH
Ar, reflux, 26 h; (iv) N O, Ar, 100 °C, 24 h; (v) 2-acetylpyridine, EtOH, Ar, 65 °C, 18 h.
ÁH
3
ÁTHF, THF, Ar, r.t., 24–72 h; (ii) glacial HOAc, reflux, 3–4 h; (iii) POCl
3
,
H
2 4
2

2
54
L.K. Filak et al. / Inorganica Chimica Acta 393 (2012) 252–260
2
4a: R1 = H, R2 = CH , M = Ru
II
II
1
1
2
2
3
3
a: R1 = CH , R = H, M = RuII
3
3
1
2
II
M
N
1
2
b: R = CH , R = H, M = Os
4b: R = H, R = CH , M = Os
3
N
3
Cl
1
2
II
1
2
II
a: R = Cl, R = H, M = Ru
b: R = Cl, R = H, M = Os
a: R = Br, R = H, M = Ru
b: R = Br, R = H, M = Os
5a: R = H, R = Cl, M = Ru
5b: R = H, R = Cl, M = Os
6a: R = H, R = Br, M = Ru
6b: R = H, R = Br, M = Os
H
Cl
1
2
II
1
2
II
N
N
1
2
II
1
2
II
1
2
II
R2
2
1
2
II
8
R1
HN
Scheme 2. Ruthenium- and osmium-arene complexes with substituted indoloquinoline ligands. (See Ref. [38] for details about 4a,b–6a,b.)
3
J = 8 Hz, 4J = 1 Hz, H ), 6.03 (d, 1H, J = 6 Hz, H ), 5.80 (d, 1H,
9
3
cy1
Found: C, 53.50; H, 4.41; N, 9.69%. ESI-MS (methanol), positive:
3J = 6 Hz, H ), 5.71 (d, 1H, 3J = 6 Hz, H ), 5.30 (d, 1H, 3J = 6 Hz,
cy2
cy1’
+
m/z 656 [MÀCl] .
cy2’
cy3
21
8a
1
11
H
2
), 2.78–2.72 (m, 1H, H ), 2.71 (s, 3H, H ), 2.54 (s, 3H, H ),
.13 (s, 3H, H ), 1.08 (d, 3H, J = 7 Hz, H ), 1.04 (d, 3H,
6
H NMR (500 MHz, DMSO-d ): 13.42 (s, 1H, H ), 10.42 (s, 1H,
cy5
3
cy4’
5
3
17
3
1
H ), 9.61 (d, 1H, J = 5 Hz, H ), 8.40 (d, 1H, J = 8 Hz, H ), 8.34 (d,
3
cy4
4
7
19
3
J = 7 Hz, H ) ppm.
1H, J = 2 Hz, H ), 8.32–8.27 (m, 1H, H ), 8.22 (d, 1H, J = 8 Hz,
1
3
, C14), 156.36 (C
, C ), 140.28 (C
, C19), 139.42 (C
20
18
3
10
C NMR (125 MHz, DMSO-d
6
): 166.18 (C
q
H
,
,
H ), 7.84–7.79 (m, 1H, H ), 7.77 (d, 1H, J = 9 Hz, H ), 7.65–
1
1
7
15
6
3
3
4
C
C
), 155.32 (C
), 137.20 (C
q
, C ), 147.54 (C
q
H
q
7.61 (m, 1H, H ), 7.59 (d, 1H, J = 8 Hz, H ), 7.49 (dd, 1H,
1a
10a
4a
8
3
3
4
9
2
q
, C ), 136.69 (C
q
, C ), 130.77 (C
q
, C ), 130.21
, C ), 124.15
, C ), 117.12
J = 9 Hz, J = 2 Hz, H ), 7.46–7.41 (m, 1H, H ), 6.06 (d, 1H,
3
, C18), 126.59 (C
20
9
J = 6 Hz, H ), 5.82 (d, 1H, J = 6 Hz, H ), 5.76 (d, 1H, 3J = 6 Hz,
cy1
3
cy2
(C
(C
(C
H
, C ), 127.34 (C
H
H
, C ), 126.54 (C
, C ), 121.91 (C
H
6
b
2
1
7
cy1‘
21
), 5.33 (d, 1H, 3J = 6 Hz, H ), 2.77–2.67 (m, 1H, H + s, 3H,
), 2.12 (s, 3H, H ), 1.07 (d, 3H, J = 7 Hz, H ), 1.04 (d, 3H,
cy2‘
cy3
q
, C ), 123.41 (C
H
, C ), 123.09 (C
, C ), 112.23 (C
, C ), 103.35 (C
H
H
H
H
4
11b
10
cy1a
cy5
3
cy4‘
H
, C ), 113.57 (C
q
H
, C ), 104.17 (C
q
, C
),
6
a
cy2a
cy1’
3
cy4
1
C
C
C
03.78 (C
q
q
, C
), 87.66 (C
, C ), 31.05 (C
, C ), 18.94 (C
H
, C ), 86.45 (C
H
,
,
,
J = 7 Hz, H ) ppm.
cy1
cy2
cy2’
cy3
13
, C14), 156.39 (C
,
H
), 86.14 (C
H
, C ), 84.70 (C
, C ), 21.78 (C
H
H
, C ), 22.25 (C
H
C NMR (125 MHz, DMSO-d
, C ), 147.23 (C
, C ), 137.45 (C
, C ), 126.76 (C
H H H H
), 124.95 (C , C ), 123.55 (C , C ), 123.42 (C , C ), 121.13 (C
6
): 166.75 (C
q
cy4
8a
cy4’
cy5
C17), 155.18 (C
15
6
11a
), 22.04 (C
) ppm.
H
H
H
, C ), 15.99 (C
H
q
q
, C ), 140.58 (C
q
, C ), 140.33
2
1
19
10a
4a
3
(C
H
q
, C ), 137.03 (C
q
, C ), 130.79 (C
H
, C ),
1
8
20
8
1
27.51 (C
H
H
, C ), 126.31 (C
q
, C ), 125.02 (C
q
,
,
6
7
b
9
2
1
C
1
2
.2.2. [Os(p-cymene)(HL )Cl]Cl, 1b
General procedure A: N-(8-Methyl-5,11-dihydroindolo[3,2-
4
10
11b
C ), 117.19 (C
H
, C ), 114.19 (C
), 103.59 (C
H
, C ), 113.28 (C
q
, C ), 104.17
cy1a
6a
cy2a
cy1’
(
C
q
, C
6.69 (C
2.33 (C
q
, C ), 103.37 (C
q
, C
, Ccy2’), 31.06 (C
H
H
), 87.45 (C , C ),
1
c]quinolin-6-ylidene)-N’-(1-pyridin-2-yl-ethylidene)azine
1
(HL ,
-chlori-
cy1
cy2
, Ccy3),
21
8
2
H
, C ), 86.11 (C
H
, C ), 84.81 (C
, Ccy4’), 18.95 (C
H H
, C ), 16.02 (C , C )
H
6
50 mg,
0.41 mmol),
bis((
g
-p-cymene)(chlorido)(
l
cy4
cy5
H
, C ), 21.70 (C
H
do)osmium(II)) (167 mg, 0.22 mmol), EtOH abs. (4 mL), CHCl
1.1 mL), diethyl ether dried over Na SO (100 mL), stirring for
8 h. Yield 207 mg, 66%. Anal. Calc. for C33 O (M
3
ppm.
(
2
4
1
7
8
H33Cl
2
N
5
OsÁ1.5H
2
r
2
2
.2.4. [Os(p-cymene)(L )Cl]Cl, 2b
87.81): C, 50.31; H, 4.61; N, 8.89. Found: C, 50.40; H, 4.45; N,
+
N-(8-Chloro-11H-indolo[3,2-c]quinolin-6-yl)-N’-(1-pyridine-2-
.87%. ESI-MS (methanol), positive: m/z 726 [MÀCl] .
6
1
11
yl-ethylidene)azine (150 mg,
0.19 mmol)
and
bis((g -p-
6
H NMR (500 MHz, DMSO-d ): 13.03 (s, 1H, H ), 9.96 (s, 1H,
5
3
17
3
1
cymene)(chlorido)(l-chlorido)osmium(II)) in a 25 mL Schlenk tube
H ), 9.54 (d, 1H, J = 6 Hz, H ), 8.36 (d, 1H, J = 8 Hz, H ), 8.32 (d,
1
7
H ), 7.45 (d, 1H, J = 8 Hz, H ), 7.42–7.38 (m, 1H, H ), 7.30 (dd,
1
3
20
19
7
were suspendend in dry ethanol (3 mL) under Ar. The mixture was
stirred for 24 h at room temperature. The precipitate formed was
collected under suction, washed with small amounts of ethanol
and dried in vacuo at 50 °C. Yield 268 mg, 88%. Anal. Calc. for
H, J = 8 Hz, H ), 8.29–8.25 (m, 1H, H ), 8.15 (s, 1H, H ), 7.82–
.78 (m, 1H, H ), 7.63 (d, 1H, J = 8 Hz, H ), 7.60–7.55 (m, 1H,
1
8
3
10
3
3
4
2
3
4
9
3
cy1
H, J = 8 Hz, J = 1 Hz, H ), 6.32 (d, 1H, J = 6 Hz, H ), 6.02 (d,
H, J = 6 Hz, H ), 5.97 (d, 1H, 3J = 6 Hz, H ), 5.43 (d, 1H,
3
cy2
cy1‘
C32H30Cl N OsÁ2.25H O (M 821.74): C, 46.77; H, 4.23; N, 8.52.
1
3
5
2
r
3
cy2‘
21
cy3
Found: C, 46.58; H, 3.83; N, 8.40%. ESI-MS (methanol), positive:
J = 6 Hz, H ), 2.78 (s, 3H, H ), 2.69–2.60 (m, 1H, H ), 2.53 (s,
+
H, H ), 2.18 (s, 3H, H ), 1.03 (d, 3H, 3J = 7 Hz, H ), 1.00 (d,
H, J = 7 Hz, H ) ppm.
8
a
cy5
cy4‘
m/z 746 [MÀCl] .
3
3
1
11
3
cy4
6
H NMR (500 MHz, DMSO-d ): 13.36 (s, 1H, H ), 10.03 (s, 1H,
5
3
17
3
1
1
3
, C14), 156.30 (C
15
H ), 9.54 (d, 1H, J = 6 Hz, H ), 8.37 (d, 1H, J = 8 Hz, H ), 8.34 (d,
C NMR (125 MHz, DMSO-d
6
): 167.18 (C
q
q
, C ),
3
20
4
7
1
7
6
19
11a
1H, J = 8 Hz, H ), 8.30 (d, 1H, J = 2 Hz, H ), 8.29–8.25 (m, 1H,
1
1
1
C
56.28 (C
37.17 (C
H q H
, C ), 147.66 (C , C ), 140.40 (C , C ), 139.35 (Cq, C ),
, C ), 136.47 (C
, C ), 126.74 (C
H H H H
), 123.38 (C , C ), 123.13 (C , C ), 121.83 (C , C ), 117.04 (C
1
9
18
3
10
1
0a
4a
8
, C3),
H ), 7.83–7.79 (m, 1H, H ), 7.76 (d, 1H, J = 9 Hz, H ), 7.63–
q
q
, C ), 130.76 (C
q H
, C ), 130.20 (C
3
3
4
9
1
8
, C20), 126.51 (C
9
7.59 (m, 1H, H ), 7.49 (dd, 1H, J = 9 Hz, J = 2 Hz, H ), 7.46 (d,
28.21 (C
H
H
H
, C ), 124.11 (C
q
,
,
,
3
4
2
3
6
4
b
2
1
7
1H, J = 8 Hz, H ), 7.44–7.40 (m, 1H, H ), 6.34 (d, 1H, J = 6 Hz,
cy1
cy1‘
cy2
3
cy2‘
11b
10
6a
H
), 6.04–6.01 (m, 2H, H
+ H ), 5.47 (d, 1H, J = 6 Hz, H ),
C ), 113.50 (C
C
q
, C ), 112.24 (C
H
, C ), 103.42 (C
q
, C ), 96.47 (C
q
2
1
cy3
cy5
cy2a
, Ccy1a), 79.44 (C
), 95.29 (C
cy1’
cy1
2.78 (s, 3H, H ), 2.67–2.59 (m, 1H, H ), 2.18 (s, 3H, H ), 1.02
(d, 3H, J = 7 Hz, H ), 1.00 (d, 3H, J = 7 Hz, H ) ppm.
q
H
, C ), 78.23 (C
H
, C ), 76.70
, C ), 22.02
, C ) ppm.
3
cy4‘
3
cy4
cy2
cy2’
cy3
cy4
(
(
C
C
H
, C ), 75.16 (C
H
, C ), 31.21 (C
H
, C ), 22.55 (C
H
1
3
14
8
a
cy4’
cy5
21
6 q H
C NMR (125 MHz, DMSO-d ): 167.77 (C , C ), 156.29 (C ,
H
, C ), 21.89 (C
H
, C ), 18.84 (C
H
, C ), 15.74 (C
H
C17), 156.17 (C
, C ), 147.35 (C , C ), 140.51 (C , C ), 140.44
q q q
15
6
11a
1
9
10a
4a
3
H q q H
(C , C ), 137.40 (C , C ), 136.83 (C , C ), 130.79 (C , C ),
2
18
20
8
6b
2
.2.3. [Ru(p-cymene)(HL )Cl]Cl, 2a
General procedure A: N-(8-Chloro-11H-indolo[3,2-c]quinolin-
-yl)-N’-(1-pyridine-2-yl-ethylidene)azine (HL , 100 mg, 0.26
128.38 (C
124.95 (C
117.14 (C
H
, C ), 126.92 (C
, C ), 123.54 (C
H
, C ), 126.32 (C
q
, C ), 125.00 (C
q
, C ),
9
2
1
7
H
H
, C ), 123.37 (C
H
, C ), 121.07 (C
H
, C ),
2
4
10
11b
6
H
, C ), 114.19 (C
H
, C ), 113.18 (C
q
, C ), 103.28 (C
q
,
,
,
6
6a
cy2a
cy1a
, Ccy1’), 78.46 (C
mmol) and bis((
g
-p-cymene)(chlorido)(
l
-chlorido)ruthenium(II))
C
C
C
q
), 96.47 (C , C
q
), 95.34 (C , C
),79.26 (C
H
H
cy1
cy2
cy2’
cy3
(
80 mg, 0.13 mmol). Yield 103 mg, 58%. Anal. Calc. for
), 76.71 (C
H
, C ), 75.31 (C
, C ), 18.85 (C
H
, C ), 31.23 (C
H
, C ), 22.62 (C
H
cy4
cy4’
cy5
21
C
32
H30Cl
3
N
5
RuÁ1.5H O (M 719.07): C, 53.45; H, 4.63; N, 9.74.
2
r
), 21.82 (C
H
H
, C ), 15.77 (C
H
, C ) ppm.

L.K. Filak et al. / Inorganica Chimica Acta 393 (2012) 252–260
255
1
3
2
.2.5. [Ru(p-cymene)(L )Cl]Cl, 3a
2.3. Characterisation in solution
General procedure A: N-(8-Bromo-11H-indolo[3,2-c]quinolin-
-yl)-N’-(1-pyridine-2-yl-ethylidene)azine (120 mg, 0.28 mmol),
1
13
1
6
bis((
0
One-dimensional H and C NMR and two-dimensional H– H
6
1
1
1
1
1
1
1
13
g
-p-cymene)(chlorido)(
l
-chlorido)ruthenium(II))
(81 mg,
COSY, H– H TOCSY, H– H ROESY or H– H NOESY, H– C HSQC
and H– C HMBC NMR spectra were recorded on two Bruker
Avance III spectrometers at 500.32 or 500.10 ( H), and 125.82 or
1
13
.13 mmol), ethanol abs. (3 mL), CHCl
over Na SO
Calc. for C32
3
(0.5 mL), diethyl ether dried
1
2
4
(180 mL), stirring for 23 h. Yield 124 mg (61%). Anal.
30BrCl O (M 763.52): C, 50.34; H, 4.36;
1
3
H
2
N
5
RuÁ1.5H
2
r
6
125.76 ( C) MHz, respectively, by using as a solvent DMSO-d or
1
N, 9.17. Found: C, 50.36; H, 3.97; N, 9.02%. ESI-MS (methanol), po-
3
CD OD at room temperature and standard pulse programs. H
+
13
sitive: m/z 702 [MÀCl] .
and C shifts are quoted relative to the solvent residual signals.
For the atom numbering scheme used for the NMR assignments,
see Chart S1. UV–Vis spectra were recorded with a Perkin Elmer
Lambda 650 spectrophotometer equipped with a six cell changer
and a Peltier element for temperature control or an Agilent 8453
spectrophotometer. Electrospray ionisation mass spectrometry
(ESI-MS) was carried out with a Bruker Esquire 3000 instrument;
the samples were dissolved in methanol. All elemental analyses
were performed at the Microanalytical Laboratory of the University
of Vienna with a Perkin Elmer 2400 CHN Elemental Analyzer. Ana-
lytical HPLC analysis was performed on a Dionex Summit system
controlled by DIONEX Chromelion 6.60 software. The experimental
conditions were as follows: a reversed phase silica-based C18 gel
1
): 13.38 (s, 1H, H11), 10.41 (s, 1H,
H NMR (500 MHz, DMSO-d
6
5
3
17
7
H ), 9.61 (d, 1H, J = 5 Hz, H ), 8.50 (br s, 1H, H ), 8.38 (d, 1H,
3
1
19
3
20
J = 8 Hz, H ), 8.32–8.26 (m, 1H, H ), 8.22 (d, 1H, J = 8 Hz, H ),
.84–7.79 (m, 1H, H ), 7.72 (d, 1H, 3J = 9 Hz, H ), 7.66–7.57 (m,
1
8
10
7
3
H
5
2
3
4
9
2
3
H, H + H + H ), 7.46–7.41 (m, 1H, H ), 6.05 (d, 1H, J = 6 Hz,
cy1
), 5.83 (d, 1H, J = 6 Hz, H ), 5.75 (d, 1H, 3J = 6 Hz, H ),
3
cy2
cy1‘
3
cy2‘
cy3
21
.34 (d, 1H, J = 6 Hz, H ), 2.77–2.66 (m, 1H, H + s, 3H, H ),
.13 (s, 3H, Hcy5), 1.07 (d, 3H, J = 7 Hz, H ), 1.04 (d, 3H,
3
cy4‘
3
cy4
J = 7 Hz, H ) ppm.
1
3
, C14), 156.38 (C
,
H
C NMR (125 MHz, DMSO-d
6
): 166.78 (C
q
C17), 155.18 (C
, C ), 147.20 (C
15
, C ), 140.39 (C
6
, C ), 140.32
11a
q
q
q
1
9
10a
4a
3
(
C
H
, C ), 137.71 (C
q
, C ), 137.04 (C
, C + C ), 126.77 (C
q
H
, C ), 130.81 (C , C ),
9
18
20
6b
1
27.52 (2 C
H
H
, C ), 125.64 (C
q
, C ), 124.17
as stationary phase (Zorbax SB-Aq, 4.6 Â 250 mm, 5
lm pore size),
7
2
1
4
(
(
C
C
H
, C ), 123.56 (C
H
, C ), 123.39 (C
, C ), 113.23 (C
, C ), 103.37 (C
H
, C ), 117.20 (C
H
, C ), 114.61
acetonitrile/15 mM aqueous formic acid as mobile phase with gra-
dient elution (5–80% acetonitrile), flow rate 1.00 mL/min, concen-
tration of the investigated complexes: 0.1 mM; 30 lL as injection
volume; 25 °C as column temperature; UV–Vis detection set at
1
0
8
11b
cy1a
H
, C ), 114.27 (C
q
q
, C ), 104.19 (C
q
, C
),
,
,
6
a
cy2a
cy1’
1
C
C
03.47 (C
), 86.11 (C
), 21.75 (C
q
q
, C
), 87.57 (C
H
, C ), 86.55 (C
, C ), 22.28 (C
, C ) ppm.
H
cy1
cy2
cy2’
cy3
H
, C ), 84.82 (C
, C ), 18.94 (C
H
, C ), 31.06 (C
, C ), 16.02 (C
H
H
cy4
cy4’
cy5
21
H
H
H
225, 254, 280 and 300 nm.
3
2
.2.6. [Os(p-cymene)(L )Cl]Cl, 3b
2.4. Crystallographic structure determination
General procedure A: N-(8-Bromo-11H-indolo[3,2-c]quinolin-
6
bis((
-yl)-N’-(1-pyridine-2-yl-ethylidene)azine (150 mg, 0.35 mmol),
X-ray diffraction measurements were performed on a Bruker X8
APEXII CCD diffractometer. Single crystals were positioned at 35,
35, 40, 35, 35 and 40 mm from the detector, and 890, 1086, 1292,
1615, 1278 and 1832 frames were measured, each for 60, 60, 50,
6
g
-p-cymene)(chlorido)(l-chlorido)osmium(II))
(137 mg,
0
.17 mmol), ethanol abs. (3 mL), CHCl
3
(0.5 mL), diethyl ether dried
over Na SO (250 mL), stirring for 23 h. The precipitate was dis-
2
4
1
4
solved in ethanol (70 mL) and solution filtered. The solvent was re-
moved under reduced pressure to ca. 5 mL and chloroform (1 mL)
was added. Then the mixture was added dropwise to diethyl ether
80, 30 and 30 s over 1° scan width for HL , HL ÁCH
3
OH,
and
O, respectively. The data were processed using SAINT soft-
5
HL Á2C
2
5
H OH
1bÁ1.38H
2
OÁ0.25(C
2 5
H )
2
O,
3aÁCH
3
OH
3bÁ0.75H
2
(
300 mL) previously dried over Na
2
SO
4
, and the precipitate formed
ware [43]. Crystal data, data collection parameters, and structure
refinement details are given in Table 1. The structures were solved
by direct methods and refined by full-matrix least-squares tech-
niques. Non-H atoms were refined with anisotropic displacement
parameters. H atoms were inserted in calculated positions and re-
fined with a riding model. The following computer programs and
hardware were used: structure solution, SHELXS-97 and refinement,
was collected under suction in vacuo at 50 °C. Further purification
was achieved by crystallisation: dissolution of crude product
(
4
100 mg) in ethanol (100 mL), evaporation of the solvent to
0 mL and slow diffusion of diethyl ether into the solution (under
Ar atmosphere). Yield: 71 mg (71%, based on 100 mg raw product
taken for the recrystallisation, overall yield 25%). Anal. Calc. for
C
32
H
30BrCl
2
N
5
OsÁ0.75H
2
O (M
r
839.17): C, 45.80; H, 3.78; N, 8.35.
SHELXL-97 [44]; molecular diagrams, ORTEP [45] computer, Intel
CoreDuo.
Found: C, 45.48; H, 3.39; N, 8.28%. ESI-MS (methanol), positive:
m/z 790 [MÀCl] .
+
1
11
6
H NMR (500 MHz, DMSO-d ): 13.26 (s, 1H, H ), 10.02 (s, 1H,
5
3
17
4
7
H ), 9.53 (d, 1H, J = 6 Hz, H ), 8.46 (d, 1H, J = 2 Hz, H ), 8.36–
2.5. Cell lines and cell culture conditions
1
20
19
8
.32 (m, 2H, H + H ), 8.30–8.25 (m, 1H, H ), 7.83–7.79 (m, 1H,
1
8
3
10
3
9
H
), 7.71 (d, 1H, J = 9 Hz, H ), 7.64–7.59 (m, 2H, H + H ), 7.46
For cytotoxicity determination, three different cell lines were
used: A549, a human non-small cell lung cancer cell line, and
SW480, a human colon carcinoma cell line (both kindly provided
by Brigitte Marian, Institute of Cancer Research, Department of
Medicine I, Medical University of Vienna, Austria) as well as CH1,
a human ovarian carcinoma cell line (kindly provided by Lloyd R.
Kelland, CRC Centre for Cancer Therapeutics, Institute of Cancer Re-
search, Sutton, UK). Cells were grown as adherent monolayer cul-
3
4
2
3
(
d, 1H, J = 8 Hz, H ), 7.45–7.40 (m, 1H, H ), 6.34 (d, 1H, J = 6 Hz,
cy1
3
cy2
3
cy1‘
H
), 6.04 (d, 1H, J = 6 Hz, H ), 6.01 (d, 1H, J = 6 Hz, H ),
3
cy2‘
21
5
.48 (d, 1H, J = 6 Hz, H ), 2.78 (s, 3H, H ), 2.68–2.61 (m, 1H,
cy3
), 2.19 (s, 3H, H ), 1.04 (d, 3H, 3J = 7 Hz, H ), 1.01 (d, 3H,
cy5
cy4‘
H
3
cy4
J = 7 Hz, H ) ppm.
1
3
, C14), 156.28 (C
, C ), 140.44 (C
, C19), 140.32 (C
q
C NMR (125 MHz, DMSO-d
6
): 167.81 (C
q
H
,
,
1
1
7
15
6
C
C
), 156.18 (C
q
, C ), 147.31 (C
q
H
1a
, C10a), 136.85 (C
4a
3
2
), 137.66 (C
q
q
, C ), 130.81 (C
H
, C ), 128.38
tures in 75 cm culture flasks (Iwaki/Asahi Technoglass) in
1
8
9
20
6b
(
C
H
, C ), 127.53 (C
H
, C ), 126.93 (C
H
, C ), 125.63 (C
q
, C ),
Minimal Essential Medium supplemented with 10% heat-inacti-
vated fetal bovine serum, 1 mM sodium pyruvate, 1% non essential
7
2
1
4
1
1
C
C
C
24.12 (C
H H H H
, C ), 123.55 (C , C ), 123.31 (C , C ), 117.17 (C , C ),
1
0
8
11b
14.60 (C
H
, C ), 114.29 (C
q
, C ), 113.14 (C
q
, C ), 103.18 (C
q
,
,
,
amino acids (from 100Â ready-to-use stock) and 4 mM
mine but without antibiotics at 37 °C under a moist atmosphere
containing 5% CO and 95% air. All cell culture media and reagents
were purchased from Sigma–Aldrich.
L-gluta-
6
a
cy2a
cy1a
cy1’
), 96.49 (C
), 76.73 (C
), 21.88 (C
q
, C
), 95.36 (C
q
, C
), 79.42 (C
H
, C ), 78.32 (C
H
cy1
cy4
cy2
cy2’
cy3
H
, C ), 75.30 (C
H
, C ), 31.23 (C
H
, C ), 22.57 (C
H
2
cy4’
, Ccy5), 15.80 (C
21
H
, C ), 18.84 (C
H
H
, C ) ppm.

2
56
L.K. Filak et al. / Inorganica Chimica Acta 393 (2012) 252–260
Table 1
1
4
5
Crystal data and details of data collection for HL , HL ÁCH
3
OH, HL Á2C
2 5
H
OH, 1bÁ1.38H
2
OÁ0.25(C
2
H
5
)
2
O, 3aÁCH
3
OH and 3bÁ0.75H
3aÁCH
34BrCl
2
O.
HL1
HL ÁCH
4
5
3bÁ0.75H
Complex
3
OH
HL Á2C
2
H
5
OH
1bÁ1.38H OÁ0.25(C
2
2
H
)
5 2
O
3
OH
2
O
Empirical formula
Forward
Space group
C
23
H
19
N
5
C
24
H
23
N
5
O
C
26
H
28ClN
477.98
P2 /n
5
O
2
C
34
H
38.25Cl
2
N
5
O
1.63Os
C
33
H
2
N
5
ORu
C
32
H
31.5BrCl N O0.75Os
2 5
365.43
P2 /n
397.47
P2 /n
804.05
P1ꢀ
768.53
P1ꢀ
839.13
P1ꢀ
1
1
1
Unit cell dimensions
a (Å)
b (Å)
c (Å)
17.0279(16)
11.1855(11)
12.0305(11)
11.4565(7)
13.1693(7)
13.3620(8)
7.4390(6)
15.6393(14)
21.2178(18)
11.2980(9)
9.3254(5)
12.3508(4)
13.3564(5)
20.5840(8)
98.018(2)
95.312(2)
112.859(3)
14.4940(12)
21.3506(18)
99.901(5)
92.072(4)
98.838(5)
3396.2(5)
4
13.5216(8)
13.5306(8)
67.646(4)
89.989(4)
85.886(3)
a
(°)
b (°)
(°)
129.439(4)
95.346(3)
99.794(5)
c
3
V (Å )
1769.6(3)
4
2007.2(2)
4
2432.5(4)
4
1573.17(16)
2
3057.97(19)
4
Z
k (Å)
0.71073
1.372
0.71073
1.315
0.71073
1.305
0.71073
1.573
0.71073
1.622
0.71073
1.823
À3
qcalcd. (g cm
)
3
Crystal size (mm )
T (K)
0.17 Â 0.15 Â 0.14
0.08 Â 0.06 Â 0.03
0.20 Â 0.13 Â 0.03
0.25 Â 0.15 Â 0.09
0.30 Â 0.20 Â 0.10
0.15 Â 0.12 Â 0.04
100(2)
100(2)
100(2)
100(2)
100(2)
150(2)
À1
l
(mm
)
0.085
0.084
0.0527
0.1427
1.029
0.190
0.0465
0.1284
1.017
3.948
0.0386
0.0988
1.057
1.974
0.0368
0.0969
1.036
5.686
0.0208
0.0506
1.018
a
R
1
0.0551
0.1610
1.004
b
wR
2
c
Goodness-of-fit (GOF)
a
b
c
R
wR
1
=
R
||F
= {
GOF = {
o
| À |F
c
||/
R
|F
o
|.
2
2
2
)2]}1/2.
2
R
[w(F
o
2 À F
c
) ]/
R
[w(F
o
2
2
2
1/2
R
[w(F
o
À F
c
) ]/(n À p)} , where n is the number of reflections and p is the total number of parameters refined.
2
.6. Cytotoxicity assay
antiproliferative activity of ruthenium- and osmium-arene com-
1
3
plexes with the ligands HL –HL reported by us previously
[38,39]. Briefly, the ligand backbone was assembled in a one-pot
reaction from substituted aminobenzylamine and isatin in glacial
acetic acid [27] (Scheme 1). The indoloquinolin-6-ones were fur-
Cytotoxicity was determined by the colorimetric MTT assay
(
MTT = 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide) as described previously [39]. Briefly, cells were harvested
by trypsinisation and seeded into 96-well plates in volumes of
1
ther chlorinated with POCl
lines with N O gave rise to the indoloquinoline-6-azines.
ÁH
Finally, condensation reaction of 2-acetylpyridine with the corre-
3
. Treatment of 6-chloro-indoloquino-
00
lL/well. Depending on the cell line, different cell densities
2
H
4
2
were used to ensure exponential growth of the untreated controls
3
3
1
3
during the experiment: 1.0 Â 10 (CH1), 2.0 Â 10 (SW480),
sponding azine afforded the chelating ligands HL –HL . For com-
3
4
3
.0 Â 10 (A549). In the first 24 h the cells were allowed to settle
parison two ligands with substituents in position 2, namely HL
5
and resume exponential growth. Then the test compounds were
dissolved in DMSO, serially diluted in medium and added to the
and HL have also been prepared and characterised (vide infra).
6
Complexes 1a,bÀ3a,b (Scheme 2) were prepared from [M(
g
-p-
II
II
plates in volumes of 100
not exceed 0.5%. After continuous exposure for 96 h (in the incuba-
tor at 37 °C and under 5% CO the medium was replaced with
00 L/well RPMI 1640 medium (supplemented with 10% heat-
inactivated fetal bovine serum and 4 mM -glutamine) and MTT
l
L/well so that the DMSO content did
cymene)Cl(
l
-Cl)]
HL –HL in absolute ethanol in 25–88% yield by exploring the
l-chlorido-bridge splitting reaction. The ESI mass spectra of the
2
, where M = Ru or Os , and indoloquinolines
1
3
2
)
,
1
l
complexes showed a single strong peak at m/z 636, 656, 702 for
the ruthenium compounds 1a–3a, and m/z 726, 746, 790 for the os-
mium congeners 1b–3b, which in all cases can be attributed to the
L
solution (MTT reagent in phosphate-buffered saline, 5 mg/mL) in
a ratio of 7:1, and plates were incubated for further 4 h. Then the
medium/MTT mixture was removed and the formed formazan
+
[MÀCl] ion.
The H and 13C NMR spectra are also consistent with the chem-
1
product was dissolved in DMSO (150
lL/well). Optical densities
ical formulae proposed for 1a,b–3a,b (Scheme 2). The number of
at 550 nm were measured (reference wavelength 690 nm) with a
microplate reader (BioTek ELx 808). The quantity of viable cells
was expressed as a percentage of untreated controls, and 50%
inhibitory concentrations (IC50) were calculated from the concen-
tration-effect curves by interpolation. Every test was repeated in
at least three independent experiments, each consisting of three
replicates per concentration level.
1
resonances indicates C point symmetry for both investigated li-
gands and complexes. All complexes are racemates due to the pres-
ence of the stereogenic metal centre. They show a typical pattern
of the four diastereotopic doublets of the aromatic p-cymene pro-
tons between 6.06 and 5.30, and between 6.34 and 5.43 ppm for
the ruthenium(II) and osmium(II) complexes, respectively.
One feature of note for this class of substances is the upfield
6
a
shift of the quaternary, aromatic carbon C to about 103 ppm in
1–3
all complexes (ca. 105 ppm for HL ), caused by the three sur-
3
. Results and discussion
5
11
12
rounding electron withdrawing nitrogen atoms N , N and N
The atom numbering scheme is depicted in Chart S1).
Most of the resonances did not shift significantly after complex-
.
(
3.1. Synthesis and characterization of organic compounds and their
metal complexes
ation. As expected, the most noteworthy changes concern the
atoms in the close proximity to the metal centre. A downfield shift
In the case of the paullones, substitution in position 9 of the li-
gand backbone led to pronounced differences both in cytotoxic and
in enzymatic inhibitory activity. Electron-withdrawing halo sub-
stituents had favourable effects on both free ligands and their me-
tal complexes [18,31]. All this prompted us to investigate the effect
of substitution in position 8 of the indoloquinoline backbone on
14
by 9–11 ppm was observed for C (166–168 ppm versus 156–
1
57 ppm in the free ligands), the shift being about 1 ppm larger
17
in the case of the osmium centre. C was shifted downfield by
about 7 ppm (156 ppm versus 149 ppm), and C –C showed up-
field shifts (4–5 ppm), again with a 1 ppm greater change for the
18
20

L.K. Filak et al. / Inorganica Chimica Acta 393 (2012) 252–260
257
osmium complexes. Other shifts in resonances were less signifi-
6
6a
21
cant, e.g. 2–3 ppm upfield shift for C , C and C . The structures
proposed for 1a,bÀ3a,b were further confirmed by X-ray
crystallography.
3.2. X-ray crystallography
1
4
The results of the X-ray diffraction studies of HL , HL ÁCH
3
OH,
5
6
1
HL Á2C
2 5
H OH, [(
g
-p-cymene)Os(L )Cl]ClÁ1.38H
2
OÁ0.25Et
2
O
(1bÁ
6
3
1
.38H
2
OÁ0.25Et
2
O), [(
g
-p-cymene)Ru(L )Cl]ClÁCH
3
OH (3aÁCH
3
OH),
6
3
[
(
g
-p-cymene)Os(L )Cl]ClÁ0.75H
2
O (3bÁ0.75H
2
O) are shown in
Fig. 1–3 and Figure S1, respectively. All three complexes crystallised
in the triclinic centrosymmetric space group P1, while the indolo-
ꢀ
1
quinoline derivatives in the monoclinic space group P2 /n. The
asymmetric units of metal complexes consist of two, one and two
crystallographically independent complexes of 1b, 3a and 3b,
respectively, and-co-crystallised solvent. The complexes have a typ-
ical ‘‘three-leg piano-stool’’ geometry of ruthenium(II) and osmiu-
Fig. 2. ORTEP view of the cation [OsCl(p-cymene)(HL
0.25(C
1
)]
+
in 1bÁ1.38H OÁ
2
6
m(II)-arene complexes, with an
g
p
-bound p-cymene ring
2
H
)
5 2
O with thermal displacement parameters drawn at 30% probability
level. Selected bond distances (Å) and angles (°): Os1ÀCl1 2.4188(14), Os1ÀN13
forming the seat and three other donor atoms (two nitrogens, N13
and N17, of indolo [3,2-c]quinoline and one chlorido ligand) as the
legs of the stool.
2
1
.095(5), Os1ÀN17 2.081(5), Os1ÀCarene(av) 2.203(9), N5ÀC6 1.365(7), C6ÀN12
.313(7), N12ÀN13 1.397(6), N13ÀC14 1.296(7) Å; N13ÀN12ÀC6ÀN5–10.6(8),
N13ÀC14ÀC16ÀN17–10.3(7).
1
The conformation adopted by the indoloquinoline ligand HL is
5
5
very close to that of HL (in HL Á2C
2 5
H OH), while being quite dif-
ferent from that found for HL4 (in HL ÁCH
4
OH), an isomer of HL
1
5
X-ray diffraction, but HL Á2C
2 5
H OH. The interplanar separation
of the indoloquinoline backbones in the pairs is of 3.393,
3
with methyl group in position 2 of the molecular backbone. This
difference is clearly seen by comparing the torsion angle
1
4
3.483, 3.448, 3.396 and 3.744 Å in HL , HL ÁCH
1bÁ1.38H OÁ 0.25(C O, 3aÁCH OH and 3bÁ0.75H O, respectively
(see Figures S2ÀS6). Figure S7 shows the packing diagram with
OH. The observed packing
peculiarities of indoloquinolines and their metal complexes
suggest, that these compounds in contrast to indolobenzazepines
possess more potential to act as DNA intercalators.
3
OH,
1
5
H
N13ÀC14ÀC16ÀN17 of 3.85(17) and 4.95(19) in HL and HL , respec-
2
H
2 5
)
2
3
2
4
tively, with that of À163.91(13)° in HL . A strong hydrogen bond-
ing interaction is evident between atom N11 acting as proton
hydrogen bonding interactions of 3aÁCH
3
i
donor and atom N17 (Àx + 0.5, yÀ0.5, Àz + 0.5) of the neighbour-
i
i
ing molecule [N11Á Á ÁN17 2.8593(15) Å, N11ÀHÁ Á ÁN17 173.0°] in
1
4
HL . In HL the atom N11 is also involved in intermolecular H-
bonding with oxygen atom of the co-crystallised methanol mole-
i
cule acting as proton acceptor. The N11ÀHÁ Á ÁO1 (xÀ1, y + 1, z + 1)
i
i
bond is also strong [N11Á Á ÁO1 2.7491(17) Å, N11ÀHÁ Á ÁO1
3.3. Optical spectra and aqueous solution behaviour
1
70.1°]. The methanol molecule also acts as a proton donor in H-
ii
1
bonding to atom N17 (x + 0.5, Ày + 0.5, zÀ0.5). The parameters
Fig. 4 depicts the UV–Vis spectra of the free ligand HL and its
Ru- and Os-complex (1a and 1b, respectively) in methanol. The
ii
ii
O1Á Á ÁN17 2.7342(18) Å, O1ÀHÁ Á ÁN17 169.2° also suggest strong
5
interaction. The conformation adopted by HL is stabilised by
free ligand exhibits strong absorptions with maxima at 229 and
⁄
intermolecular hydrogen bonding interactions with an ethanol
molecule as shown in Figure S1.
Another feature of note is the presence of isolated pairs of mol-
ecules or complex cations with offset parallel arrangement stabi-
265 nm due to intraligand
p
Àp
transitions. Upon complexation
to Ru and Os-arene moieties a strong band at 298 and 303 nm,
respectively, appeared. In addition another absorption in the visi-
ble region with maximum between 450 and 500 nm is seen, which
can presumably be attributed to metal-to-ligand charge transfer.
lised by
p-stacking interactions in all compounds studied by
Fig. 1. ORTEP view of HL1 (left) and HL4 (right) with thermal displacement parameters drawn at 50% probability level. Selected bond distances (Å) and angles (°) for HL :
1
4
N5ÀC6 1.3718(16), C6ÀN12 1.3201(16), N12ÀN13 1.3874(14), N13ÀC14 1.2913(16) Å; N13ÀN12ÀC6ÀN5 6.14(16), N13ÀC14ÀC16ÀN17 3.85(17); for HL : N5ÀC6 1.369(2),
C6ÀN12 1.3238(19), N12ÀN13 1.3839(18), N13ÀC14 1.2969(19) Å; N13ÀN12ÀC6ÀN5–0.3(2), N13ÀC14ÀC16ÀN17–163.91(13).

2
58
L.K. Filak et al. / Inorganica Chimica Acta 393 (2012) 252–260
3
+
3
+
Fig. 3. ORTEP views of the cation [RuCl(p-cymene)(HL )] in 3aÁCH
3
OH (left) and of the first crystallographically independent cation [OsCl(p-cymene)(HL )] in 3bÁ0.75H
2
O
(
right) with thermal displacement parameters drawn at 50% and 30% probability level, respectively. Selected bond distances (Å) and angles (°): RuÀCl1 2.3965(7), RuÀN13
2
.084(2), RuÀN17 2.089(2), RuÀCarene(av) 2.208(11), N5ÀC6 1.368(3), C6ÀN12 1.320(3), N12ÀN13 1.409(3), N13ÀC14 1.304(3) Å; N13ÀN12ÀC6ÀN5–2.7(4),
N13ÀC14ÀC16ÀN17 5.4(3); Os1ÀCl1 2.4049(8), Os1ÀN13 2.097(2), Os1ÀN17 2.072(2), RuÀCarene(av) 2.187(12), N5ÀC6 1.366(4), C6ÀN12 1.314(4), N12ÀN13 1.398(3),
N13ÀC14 1.303(4) Å; N13ÀN12ÀC6ÀN5–14.0(4), N13ÀC14ÀC16ÀN17–10.9(4).
The aqueous solution behaviour of ruthenium and osmium com-
plexes with respect to hydrolysis was studied at 298 K over 24 h by
UV–Vis spectroscopy. The osmium complexes 1bÀ3b are quite sta-
ble in aqueous solution with 1% DMSO, while ruthenium congeners
microculture assay (MTT assay) was used in three human cancer
À7
cell lines (A549, CH1, SW480), yielding IC50 values in the 10
–
À6
10 M range after continuous exposure for 96 h (Table 2). A549,
a generally more chemoresistant cell line, is the least sensitive to
all the tested compounds, whereas in CH1 and SW480 cells up to
one order of magnitude lower IC50 values were found. The uncom-
plexed ligands could not be tested because of their insufficient
solubility.
(
1aÀ3a) undergo hydrolysis to a certain extent (Figure S8). To fur-
ther investigate the nature of changes observed in UV–Vis spectra
the experiment was repeated by using higher concentrations of
6 2
ruthenium complex in 10% DMSO-d in D O as a solvent and the
process was monitored by 1H NMR spectroscopy. There were no
signs of decomposition or release of ligands after 24 h, possibly ow-
ing to the much higher concentration of the complex in the NMR
Comparison of the ruthenium(II) with their osmium(II) ana-
logues shows up to five times higher activity of the former in
SW480 cells, whereas in CH1 and A549 cells all IC50 values are in
a comparable range. The different substituents, methyl (1a, 1b),
chloro (2a, 2b) or bromo (3a, 3b) in position 8, have no pronounced
(if any) effect on cytotoxicity of the complexes (Table 2, Fig. 5).
This is in accordance with structure–activity relationships of re-
lated copper(II) complexes with indoloquinolines reported previ-
ously [39], showing that major effects on cytotoxicity were only
observed for the presence/absence of a methyl group at c14 in
the ligand side chain, which is invariably methylated in all the
ruthenium(II) and osmium(II) complexes studied here. The cop-
per(II) complexes of the ligands methylated at c14 are 10–50 times
more cytotoxic in all tested cell lines. Related ruthenium(II) and
tube (ca 2 mM compared to 35–40
Partial dissociation of the ruthenium(II) complexes can be expected
in case of not very high thermodynamic stability) with decreasing
lM in the UV–Vis experiment).
(
analytical concentrations. Therefore, the aqueous behaviour was
further investigated by analytical HPLC. The results for complexes
3
a and 3b are illustrated in Figure S9. Whereas the initial ruthe-
nium complex underwent at least two minor reactions, the osmium
complex 3b remained intact over 24 h, what is typical for other os-
mium(II)-arene complexes [37,46,47], which are markedly more in-
ert compared to ruthenium congeners.
3.4. Cytotoxicity in cancer cells
To determine the cytotoxicity of the ruthenium(II)- and os-
Table 2
Comparison of cytotoxicity of ruthenium(II)- and osmium(II)-arene complexes with
modified indoloquinoline ligands 1a,bÀ3a,b vs. 4a,bÀ6a,b (reported previously [38])
in three human cancer cell lines.
mium(II)-arene complexes of indoloquinolines, a colorimetric
Compound
IC50a
(lM), 96 h
A549
SW480
CH1
1
1
2
2
3
3
4
4
5
5
6
6
a
b
a
b
a
b
a
b
a
b
a
b
2.3 ± 0.7
1.9 ± 0.2
2.2 ± 0.7
2.5 ± 0.4
1.6 ± 0.3
2.0 ± 0.2
2.0 ± 0.4
3.2 ± 0.4
9.3 ± 3.4
3.9 ± 0.5
7.2 ± 1.7
7.8 ± 2.1
7.1 ± 1.1
0.13 ± 0.03
0.67 ± 0.14
0.38 ± 0.06
0.83 ± 0.24
0.33 ± 0.04
0.44 ± 0.15
0.28 ± 0.02
0.57 ± 0.20
5.0 ± 1.0
1.2 ± 0.3
1.5 ± 0.6
2.3 ± 0.4
3.5 ± 0.5
0.22 ± 0.03
0.22 ± 0.01
0.34 ± 0.09
0.23 ± 0.04
0.20 ± 0.05
0.20 ± 0.03
0.19 ± 0.02
0.19 ± 0.08
3.8 ± 0.6
0.55 ± 0.14
1.3 ± 0.2
1.0 ± 0.4
[g
6-p-cymene)Ru(II)(en)Cl](PF
6
)b
4.4 ± 0.9
a
Fig. 4. UV–Vis spectra of HL1 (37
36 M, blue line) and 1b (39 M, red line) in methanol. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of
50% Inhibitory concentrations (means ± standard deviations from at least three
independent experiments), as obtained by the MTT assay using exposure times of
6 h.
l
M, green line) and its Ru- and Os complex 1a
(
l
l
9
b
Taken from Ref. [48].
this article.)

L.K. Filak et al. / Inorganica Chimica Acta 393 (2012) 252–260
259
compounds were comprehensively characterised by 1D and 2D
NMR techniques, ESI mass spectrometry, optical spectroscopy
and X-ray crystallography (three ligands and three complexes).
Complexation of indoloquinoline ligands with ruthenium(II) or
osmium(II) resulted in improved solubility in biological media
enabling biological assays. Substitution in position 8 seems to be
more favourable for the cytotoxic activity than that in position 2,
at least for the halogenated indoloquinoline complexes. Especially
the most effective compounds with IC50 values in the submicrom-
olar concentration range are worth being studied for their suitabil-
ity as potential anticancer drugs.
Acknowledgements
We thank R. Bugl for assistance with the synthesis of indolo-
quinolines, A. Luganschi and A. Dobrov for ESI-MS measurements,
Prof. M. Galanski for the measurement of the 2D NMR spectra,
Alexander Roller for collection of X-ray data, as well as V. Pichler
and R. Bugl for the HPLC chromatograms. Financial support of
the FWF (Austrian Science Fund, Project number P22339) is kindly
acknowledged.
Appendix A. Supplementary data
CCDC 878664–878669 contains the supplementary crystallo-
graphic 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. Supplementary data associ-
ated with this article can be found, in the online version, at http://
dx.doi.org/10.1016/j.ica.2012.06.004.
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