Organometallic complexes of (thio)allomaltol-based Mannich-products: Synthesis, stability and preliminary biological investigations

Article abstract of DOI:10.1016/j.jorganchem.2014.10.044

Abstract Organometallic complexes with (thio)pyrone-based ligands have been shown to possess promising cytotoxic properties. To extend the class of potential metallodrugs, the (thio)pyrone backbone was modified via Mannich reaction with morpholine, N-methylpiperazine and piperidine as cyclic amine. The ligands and organometallic complexes were characterized by means of 1D and 2D NMR, ESI MS and also in one case by X-ray diffraction analysis. Due to the high aqueous solubility, the behavior and stability in aqueous solution of the synthesized complexes was studied by ¹H NMR spectroscopy. In addition, the influence of these modifications on cytotoxicity in human cancer cell lines was investigated by means of the MTT assay.

Full text of DOI:10.1016/j.jorganchem.2014.10.044

Journal of Organometallic Chemistry xxx (2014) 1e8
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
Organometallic complexes of (thio)allomaltol-based Mannich-
products: Synthesis, stability and preliminary biological investigations
,
b
Melanie Schmidlehner a, Verena Pichler a, Alexander Roller a, Michael A. Jakupec a
,
,
b
,
, b
Wolfgang Kandioller a *, Bernhard K. Keppler a
a Institute of Inorganic Chemistry, Faculty of Chemistry, University of Vienna, Waehringer Str. 42, A-1090 Vienna, Austria
b University of Vienna, Research Platform “Translational Cancer Therapy Research”, Waehringer Str. 42, A-1090 Vienna, Austria
a r t i c l e i n f o
a b s t r a c t
Article history:
Organometallic complexes with (thio)pyrone-based ligands have been shown to possess promising
cytotoxic properties. To extend the class of potential metallodrugs, the (thio)pyrone backbone was
modified via Mannich reaction with morpholine, N-methylpiperazine and piperidine as cyclic amine. The
ligands and organometallic complexes were characterized by means of 1D and 2D NMR, ESI MS and also
in one case by X-ray diffraction analysis. Due to the high aqueous solubility, the behavior and stability in
aqueous solution of the synthesized complexes was studied by 1H NMR spectroscopy. In addition, the
influence of these modifications on cytotoxicity in human cancer cell lines was investigated by means of
the MTT assay.
Received 26 September 2014
Received in revised form
29 October 2014
Accepted 30 October 2014
Available online xxx
Keywords:
Organometallic chemistry
Ruthenium
© 2014 Elsevier B.V. All rights reserved.
Rhodium
Half-sandwich complexes
Pyrones
Thiopyrones
Introduction
facial coordination of an arene moiety, yielding compounds with a
piano-stool geometry of the general formula [(h
6-arene)Ru(X,Y,Z)],
Ruthenium complexes are promising agents for the treatment of
cancer and the most prominent representatives of this class of
metallodrugs, sodium trans-[tetrachloridobis(1H-indazole)ruth-
enate(III)] (NKP-1339 or IT-139) and imidazolium trans-[tetra-
where X,Y are monodentates or a chelating moiety and Z is the
leaving group, typically a halido ligand. With variation of the
remaining coordination sites, chemical reactivity and anticancer
activity can be fine-tuned, notably bidentate chelating ligands
showed a high impact on the stability [6e8]. The hydrophobic ar-
ene may enhance the transport through the cell membrane and
facilitate cellular uptake [9]. The two most investigated represen-
tatives of this class so far are the RAPTA compounds (e.g. RAPTA-C),
developed by Dyson and coworkers, and 1,2-diaminoethane com-
plexes established by Sadler's group (e.g. RM175). Both paved the
way for further investigation of organometallic Ru(II)-arene com-
pounds as potential anticancer agents and are in an advanced
preclinical stage of drug development [6,10e13].
3-Hydroxypyrones are widely used as chelating agents due to
their ability to form stable complexes with a broad range of
different metal ions. Advantages include the easy accessibility, high
biocompatibility and the high amount of possible modifications.
Recently, metal complexes bearing 3-hydroxy(thio)pyrones as a
chelating motif have been shown to possess promising cytotoxic
activity in vitro and are currently being tested in vivo [14e16]. These
neutral complexes undergo rapid hydrolysis within seconds,
yielding the respective aqua species. It has been found that
chlorido(1H-imidazole)(dimethylsulfoxide-kS)ruthenate(III)]
(NAMI-A), have been investigated in clinical trials (Fig. 1) [1e4].
NKP-1339 is administered intravenously and binds rapidly to blood
proteins such as albumin and transferrin. The formed adducts
prevent the complex from fast aquation and decomposition in
aqueous solution and are then accumulated in the tumor tissue.
There the metal center is reduced under hypoxic conditions (acti-
vation by reduction), yielding the corresponding highly reactive
Ru(II) species, which are thought to interact with cellular targets
[3,5].
Due to the supposed reduction of the clinically tested Ru(III)
metallodrugs under hypoxic conditions, the class of Ru(II) com-
plexes has been investigated for anticancer potential as well. Ru(II)
is prone to oxidation reactions, but it can be easily stabilized by
* Corresponding author. Institute of Inorganic Chemistry, Faculty of Chemistry,
University of Vienna, Waehringer Str. 42, A-1090 Vienna, Austria. Tel.: þ43 1 4277
52609; fax: þ43 1 4277 9526.
E-mail address: wolfgang.kandioller@univie.ac.at (W. Kandioller).
http://dx.doi.org/10.1016/j.jorganchem.2014.10.044
0022-328X/© 2014 Elsevier B.V. All rights reserved.
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j.jorganchem.2014.10.044

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M. Schmidlehner et al. / Journal of Organometallic Chemistry xxx (2014) 1e8
Fig. 1. Structures of ruthenium anticancer agents investigated in clinical trials (NAMI-A and NKP-1339) or in an advanced preclinical stage of drug development (RAPTA-C and
RM175).
thiopyrone analogs are significantly more stable in the presence of
biomolecules than the analogous pyrone derivatives, where ligand
cleavage was observed over time [15,17]. The increase in stability is
supposed to be the crucial factor for the observed high cytotoxic
activity in the low micromolar range [15,17,18].
for 4 h under argon atmosphere. After cooling to room tempera-
ture the solvent was evaporated and the crude product was pu-
rified by column chromatography (n-hexane/ethyl acetate ¼ 10:1,
Rf ¼ 0.15). Yield: 80%. 1H NMR (DMSO-d6, 500.10 MHz, 25 ꢀC):
d
¼ 2.32 (s, 3H, CH3), 7.33 (s, 1H, H3), 8.25 (s, 1H, H6), 8.41 (s, 1H,
Due to the lower affinity of oxygen to ruthenium, O,O-bidentate
ligands tend to be cleaved off after dissolving in water or in the
presence of biomolecules. Kasser and coworkers have already re-
ported a series of organometallic Mannich products of kojic acid.
The utilization of kojic acid as precursor resulted in a high solubi-
lity; however, the stability under biological conditions was limited
due to the formation of [Ru2(p-cym)2(OH)3]þ after a short period of
time [19]. Utilization of thiopyrones might circumvent this draw-
back, and the aim of this work was the extension of the (thio)
pyrone library by modification of the heterocyclic backbone via
Mannich reaction and subsequent conversion to corresponding
Ru(II)-, Os(II)-arene and Rh(II)-Cp* complexes. The solubility and
stability of the novel compounds in water was examined. The
impact of the metal center variation and the introduction of hy-
drophilic groups on the anticancer activity was elucidated by the
colorimetric MTT assay in different human cancer cell lines.
eOH) ppm.
General procedure for Mannich reactions
A mixture of cyclic secondary amine (1.30 mmol, 0.8 eq.) and
aqueous formaldehyde solution (35%, 0.127 mL, 1.59 mmol, 1 eq.) in
methanol (6 mL) was heated to reflux temperature. Allomaltol
(200 mg, 1.59 mmol, 1 eq.) or thioallomaltol (203 mg, 1.586 mmol,
1 eq.) was added and the mixture was refluxed for a further 5 min.
After cooling to room temperature, the solution was concentrated
under reduced pressure and the formed precipitate was separated
by filtration, washed with petrol ether and dried in vacuo.
3-Hydroxy-6-methyl-2-[(morpholin-4-yl)methyl]-pyran-4(1H)-one
(3a)
The reaction was performed according to the general procedure
using morpholine (113 mL, 113 mg) and allomaltol (200 mg). Yield:
Experimental
83%, colorless solid. 1H NMR (DMSO-d6, 500.10 MHz, 25 ꢀC):
3
d
¼ 2.25 (s, 3H, CH3), 2.43 (t, JH,H ¼ 5 Hz, 4H, Hmor), 3.48 (s, 2H,
General
CH2eN), 3.56 (t, 3JH,H ¼ 5 Hz, 4H, Hmor), 6.22 (s, 1H, H5), 8.85 (br s,
1H, eOH) ppm. 13C NMR (DMSO-d6, 127.75 MHz, 25 ꢀC):
d
¼ 173.5
All solvents were of analytical grade and used without further
purification. Utilized chemicals were purchased from Sigma Aldrich
or Acros and used as received. The dimeric metal precursors [Ru(p-
cym)Cl2]2, [Os(p-cym)Cl2]2, and [Rh(Cp*)Cl2]2 were prepared ac-
cording to the literature [20,21]. Allomaltol was synthesized ac-
cording to literature protocols. The synthesis of thioallomaltol was
slightly modified, compared to the standard procedure [15,22e24].
1H NMR spectra were recorded at 25 ꢀC and 500.10 MHz and 13C{H}
NMR spectra at 25 ꢀC and 125.75 MHz using a Bruker FT-NMR
spectrometer Avance III™ 500 MHz. D2O, DMSO-d6 and CDCl3
were used as solvents for the NMR experiments and the concen-
trations of the samples were around 2e4 mM. Electrospray ioni-
zation mass spectra were recorded on a Bruker Esquire 3000 in
positive and negative mode; the samples were dissolved in meth-
anol. Elemental analyses were carried out with a Eurovector
EA3000 Elemental Analyzer by the Microanalytical Laboratory of
the University of Vienna. If not otherwise stated, the substances
were synthesized and purified according to general procedures.
(C4), 164.7 (C6), 146.2 (C2), 143.4 (C3), 111.1 (C5), 66.1 (CH2mor) 53.9
(CH2eN), 53.0 (CH2mor), 19.4 (C7) ppm. Elemental Anal. Calc for
C
11H15NO4: C, 58.66; H, 6.71; N, 6.22; Found: C, 58.56; H, 6.51; N,
6.04%. ESI-MS (neg) m/z: 223.4 [M ꢁ H]ꢁ. ESI-MSþ m/z: 225.6
[M þ H]þ, 247.5 [M þ Na]þ, 472.4 [2M þ H]þ.
3-Hydroxy-6-methyl-2-[(piperidin-1-yl)methyl]-pyran-4(1H)-one
(3b)
The reaction was performed according to the general procedure
using piperidine (128 mL, 110 mg) and allomaltol (200 mg). After
standard work up, the product was obtained as highly viscous oil.
Therefore, the oil was dissolved in dichloromethane and concen-
trated to dryness, yielding the compound as a colorless solid. Yield:
88%, colorless solid. 1H NMR (DMSO-d6, 500.10 MHz, 25 ꢀC):
d
¼ 1.33e1.38 (m, 2H, Hpip),1.46e1.50 (m, 4H, Hpip) 2.25 (s, 3H, CH3),
2.39e2.41 (m, 4H, Hpip), 3.45 (s, 2H, CH2eN), 6.20 (s, 1H, H5), 8.88
(br s, 1H, eOH) ppm. 13C NMR (DMSO-d6, 127.75 MHz, 25 ꢀC):
d
¼ 173.4 (C4), 164.5 (C6), 146.8 (C2), 143.2 (C3), 111.1 (C5), 54.4
Synthesis
(CH2eN), 53.7 (CH2pip), 25.4 (CH2pip), 23.6 (CH2pip), 19.3 (C7) ppm.
Elemental Anal. Calc for C12H17NO3 $ 0.1CH2Cl2: C, 62.71; H, 7.48; N,
6.04; Found: C, 62.69; H, 7.27; N, 5.78%. ESI-MSꢁ m/z: 221.4
[M ꢁ H]ꢁ. ESI-MSþ m/z: 223.7 [M þ H]þ, 446.4 [2M þ H]þ, 468.5
[2M þ Na]þ.
5-Hydroxy-2-methyl-thiopyran-4(1H)-one (2)
Allomaltol (1.00 g, 7.9 mmol) and Lawesson's reagent (1.61 g,
4.0 mmol) were suspended in 20 mL dry 1,4-dioxane and refluxed
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M. Schmidlehner et al. / Journal of Organometallic Chemistry xxx (2014) 1e8
3
3-Hydroxy-6-methyl-2-[(4-methylpiperazin-1-yl)methyl]-pyran-
4(1H)-one (3c)
Chlorido{3-(oxo-
k
O)-6-methyl-2-[(piperidin-1-yl)methyl]pyran-
4(1H)-onato- O}(
k
h
6-p-cymene)ruthenium(II) (4b).
The reaction was performed according to the general procedure
The reaction was performed analogous to the general
complexation procedure, using 3b (119 mg, 0.53 mmol), sodium
methoxide (29 mg, 0.53 mmol) and [Ru(p-cym)Cl2]2 (150 mg,
0.25 mmol). Yield: 73%.
using N-methylpiperazine (144
(200 mg). Yield: 92%, colorless solid. 1H NMR (DMSO-d6,
500.10 MHz, 25 ꢀC):
mL, 130 mg) and allomaltol
d
¼ 2.15 (s, 1H, NeCH3), 2.26 (s, 3H, CH3), 2.31
(br s, 4H, Hpaz), 2.45 (br s, 4H, Hpaz) 3.49 (s, 2H, CH2eN), 6.22 (s, 1H,
1H NMR (CDCl3, 500.10 MHz, 25 ꢀC):
d
¼ 1.28 (d, 3JH,H ¼ 7 Hz, 3H,
H5), 8.89 (br s, 1H, eOH) ppm. . 13C NMR (DMSO-d6, 127.75 MHz,
CH3cym-c), 1.33 (d, 3JH,H ¼ 7 Hz, 3H, CH3cym-c), 1.43 (br s, 2H, CH2pip),
1.59 (br s, 4H, CH2pip), 2.25 (s, 3H, CH3), 2.30 (s, 3H, CH3cym-a),
2.50e2.56 (m, 4H, Hpip), 2.89 (sept, 3JH,H ¼ 7 Hz, 1H, CHcym-b), 3.45
25 ꢀC):
d
¼ 173.5 (C4), 164.6 (C6), 146.6 (C2), 143.2 (C3), 111.1 (C5),
54.4 (CH2pip), 53.5 (CH2eN), 52.3 (CH2pip), 45.6 (NeCH3), 19.4 (C7)
ppm. Elemental Anal. Calc for C12H18N2O3 $0.15CH3OH: C, 60.03; H,
7.71; N, 11.52; Found: C, 60.26; H, 7.37; N, 11.20%. ESI-MSꢁ m/z:
236.5 [M ꢁ H]ꢁ. ESI-MSþ m/z: 238.7 [M þ H]þ, 476.5 [2M þ H]þ,
498.5 [2M þ Na]þ.
2
(br s, 1H, CH2eN), 3.89 (d, JH,H ¼ 13 Hz, 1H, CH2eN), 5.23 (d,
3
3JH,H ¼ 6 Hz, 1H, Hcym), 5.28 (d, JH,H ¼ 6 Hz, 1H, Hcym), 5.47 (d,
3JH,H ¼ 6 Hz, 1H, Hcym), 5.47 (d, 3JH,H ¼ 6 Hz, 1H, Hcym), 6.27 (s, 1H,
H5) ppm. 13C NMR (CDCl3, 127.75 MHz, 25 ꢀC):
d
¼ 184.8 (C4), 164.3
(6), 109.5 (C5), 99.6 (Ccym-4), 96.1 (Ccym-1), 80.1 (Ccym-3), 79.9 (Ccym-
5), 87.2 (Ccym-2,6), 54.6 (NeCH3), 54.4 (CH2pip), 31.2 (CHcym-b), 26.0
(CH2pip), 24.2 (CH2pip), 22.7 (CH3cym-c), 22.3 (CH3cym-c), 20.2 (C7),
18.7 (CH3cym-a) ppm. Elemental Anal. Calc for C22H30ClNO3Ru: C,
53.60; H, 6.13; N, 2.84; Found: C, 53.40; H, 5.90; N, 2.74%. ESI-MSþ
m/z: 457.5 [M ꢁ Cl]þ, 373.5 [M ꢁ Cl ꢁ Mpip]þ.
3-Hydroxy-6-methyl-2-[(morpholin-4-yl)methyl]-thiopyran-4(1H)-
one (3d)
The reaction was performed according to the general procedure
using thioallomaltol (203 mg) and morpholine (113 mL, 113 mg).
After removal of the solvent, the oily residue was dissolved in
diethyl ether and stored overnight at 4 ꢀC. The precipitate was
collected, washed with diethyl ether and dried in vacuo. Yield: 73%,
Chlorido{3-(oxo-
pyran-4(1H)-onato-
k
O)-6-methyl-2-[(4-methylpiperazin-1-yl)methyl]
O}(
6-p-cymene)ruthenium(II) (4c).
brown solid. 1H NMR (DMSO-d6, 500.10 MHz, 25 ꢀC):
d
¼ 2.34 (s, 3H,
k
h
CH3), 2.46 (t, 3JH,H ¼ 4 Hz, 4H, Hmor), 3.57 (t, 3JH,H ¼ 4 Hz, 4H, Hmor),
The reaction was performed analogous to the general
complexation procedure, using 3c (128 mg, 0.53 mmol), sodium
methoxide (29 mg, 0.53 mmol) and [Ru(p-cym)Cl2]2 (150 mg,
0.25 mmol). Yield: 66%
3.61 (s, 2H, CH2eN), 7.32 (s, 1H, H5), 8.31 (br s, 1H, eOH) ppm. 13
C
NMR (DMSO-d6, 127.75 MHz, 25 ꢀC):
d
¼ 187.5 (C4), 159.5 (C6),
149.37 (C3), 143.2 (C2), 123.1 (C5), 66.1 (CH2mor) 54.4 (CH2eN), 53.0
(CH2mor), 18.8 (C7) ppm. Elemental Anal. Calc for C11H15NO3S: C,
54.75; H, 6.27; N, 5.80; S, 13.29 Found: C, 54.54; H, 6.23; N, 5.62; S,
13.29%. ESI-MSꢁ m/z: 239.4 [M ꢁ H]ꢁ. ESI-MSþ m/z: 241.6 [M þ H]þ,
263.6 [M þ Na]þ
1H NMR (CDCl3, 500.10 MHz, 25 ꢀC):
d
¼ 1.28 (d, 3JH,H ¼ 7 Hz, 3H,
CH3cym-c), 1.33 (d, 3JH,H ¼ 7 Hz, 3H, CHeCH3cym-c), 2.25 (s, 3H, CH3),
2.30 (s, 3H, NeCH3), 2.30 (s, 3H, CH3cym-a), 2.47 (br s, 4H, Hpaz), 2.63
3
(br s, 4H, Hpaz), 2.89 (sept, JH,H ¼ 7 Hz, 1H, CHcym-b), 3.46 (d,
2JH,H ¼ 13 Hz, 1H, CH2eN), 3.96 (d, 2JH,H ¼ 13 Hz, 1H, CH2eN), 5.24
3
(d, 3JH,H ¼ 6 Hz, 1H, Hcym), 5.28 (d, JH,H ¼ 6 Hz, 1H, Hcym), 5.47 (d,
Synthesis of the ruthenium(II) and rhodium(III) complexes
3JH,H ¼ 6 Hz, 1H, Hcym), 5.49 (d, 3JH,H ¼ 6 Hz, 1H, Hcym), 6.27 (s, 1H,
General procedure for the synthesis of pyrone-based organometallic
complexes. Ligand (1.1 eq) and sodium methoxide (1.1 eq) were
dissolved in methanol (10 mL) and stirred for 15 min under argon
atmosphere. The respective dimeric metal precursor (150 mg, 1 eq)
was added to the solution and the mixture was stirred overnight.
The solvent was removed under reduced pressure, dissolved in
dichloromethane and filtered. The volume was reduced to ~2 mL, n-
hexane was added and the suspension was stored overnight at 4 ꢀC.
The formed solid was separated by filtration, washed with n-hex-
ane and dried in vacuo.
H5) ppm. 13C NMR (CDCl3, 127.75 MHz, 25 ꢀC):
d
¼ 185.0 (C4), 164.4
(C6), 158.2 (C3), 149.9 (C2), 109.5 (C5), 99.6 (Ccym-4), 96.0(Ccym-1),
80.2 (Ccym-3), 79.8 (Ccym-5), 87.4 (Ccym-2,6), 55.2 (CH2paz), 53.8
(CH2paz), 52.8 (CH2eN), 46.1 (NeCH3), 31.2 (CHcym-b), 22.7 (CH3cym-
c), 22.4 (CH3cym-c), 20.22 (C7),18.74 (CH3cym-a) ppm. Elemental Anal.
Calc for C22H31ClN2O3Ru$0.5H2O: C, 51.11; H, 6.24; N, 5.42; Found:
C, 51.07; H, 5.91; N, 5.23%. ESI-MSþ m/z: 472.5 [M ꢁ Cl]þ, 373.5
[M ꢁ Cl ꢁ Mpip]þ.
Chlorido{3-(oxo-
4(1H)-onato-kO}(h
k
O)-6-methyl-2-[(morpholin-4-yl)methyl]pyran-
5-1,2,3,4,5-pentamethylcyclopentadienyl)rhodiu-
Chlorido{3-(oxo-
4(1H)-onato- O}(
k
O)-6-methyl-2-[(morpholin-4-yl)methyl]pyran-
6-p-cymene)ruthenium(II) (4a).
m(III) (6a).
k
h
The reaction was performed analogous to the general
complexation procedure, using 3a (120 mg, 0.53 mmol), sodium
methoxide (29 mg, 0.53 mmol) and [Rh(Cp*)Cl2]2 (150 mg,
0.24 mmol). Yield: 64%.
The reaction was performed analogous to the general
complexation procedure, using 3a (120 mg, 0.53 mmol), sodium
methoxide (29 mg, 0.53 mmol) and [Ru(p-cym)Cl2]2 (150 mg,
0.25 mmol). Yield: 66%.
1H NMR (CDCl3, 500.10 MHz, 25 ꢀC):
d
¼ 1.70 (s, 15H, Cp*), 2.24
1H NMR (CDCl3, 500.10 MHz, 25 ꢀC):
d
¼ 1.29 (d, 3JH,H ¼ 7 Hz, 3H,
(s, 3H, CH3), 2.62 (br s, 4H, Hmor), 3.39 (d, 2JH,H ¼ 14 Hz, 1H, CH2eN),
CH3cym-c), 1.33 (d, 3JH,H ¼ 7 Hz, 3H, CH3cym-c), 2.26 (s, 3H, CH3), 2.30
3
3.69e3.76 (m, 4H, Hmor), 4.03 (d, 2JH,H ¼ 14 Hz, 1H, CH2eN), 6.25 (s,
(s, 3H, CH3cym-a), 2.54e2.63 (m, 4H, Hmor), 2.90 (sept, JH,H ¼ 7 Hz,
1H, H5) ppm. 13C NMR (CDCl3, 127.75 MHz, 25 ꢀC):
d
¼ 184.6 (C4),
1H, CHcym-b), 3.48 (d, 2JH,H ¼ 13 Hz, 1H, CH2eN), 3.71 (t, 3JH,H ¼ 5 Hz,
4H, Hmor), 3.85 (d, 2JH,H ¼ 13 Hz, 1H, CH2eN), 5.24 (d, 3JH,H ¼ 6 Hz,
1H, Hcym), 5.28 (d, 3JH,H ¼ 6 Hz, 1H, Hcym), 5.48 (d, 3JH,H ¼ 6 Hz, 1H,
163.7 (C6), 158.4 (C3), 149.2 (C2), 110.1 (C5), 91.2 (Cp*), 67.2
(CH2mor), 54.5 (CH2eN), 53.5 (CH2mor), 20.2 (C7), 9.02 (Cp*) ppm.
Elemental Anal. Calc for C21H29ClNO4Rh: C, 50.67; H, 5.87; N, 2.81;
Found: C, 50.76; H, 5.84; N, 2.62%. ESI-MSþ m/z: 461.6 [M ꢁ Cl]þ.
H
cym), 5.50 (d, 3JH,H ¼ 6 Hz,1H, H cym), 6.28 (s,1H, H5) ppm. 13C NMR
(CDCl3, 127.75 MHz, 25 ꢀC):
d
¼ 185.1 (C4), 164.5 (C6), 158.2 (C3),
149.4 (C2), 109.5 (C5), 99.5 (Ccym-4), 95.93 (Ccym-1), 80.2 (Ccym-3),
80.0 (Ccym-5), 78.2 (Ccym-2), 77.5 (Ccym-6), 67.0 (CH2mor), 54.4
(CH2eN), 53.5 (CH2mor), 31.2 (CHcym-b), 22.6 (CH3cym-c), 22.4
(CH3cym-c), 20.2 (C7), 18.7 (CH3cym-a) ppm. Elemental Anal. Calc for
Chlorido{3-(oxo-
4(1H)-onato-kO}(h
m(III) (6b).
k
O)-6-methyl-2-[(piperidin-1-yl)methyl]pyran-
5-1,2,3,4,5-pentamethylcyclopentadienyl)rhodiu-
C
21H28ClNO4Ru: C, 50.96; H, 5.70; N, 2.83; Found: C, 50.66; H, 5.36;
The reaction was performed analogous to the general
complexation procedure, using 3b (119 mg, 0.53 mmol), sodium
N, 2.79%. ESI-MSþ m/z: 459.5 [M ꢁ Cl]þ, 373.5 [M ꢁ Cl ꢁ Mmor]þ.
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j.jorganchem.2014.10.044

4
M. Schmidlehner et al. / Journal of Organometallic Chemistry xxx (2014) 1e8
methoxide (29 mg, 0.53 mmol) and [Rh(Cp*)Cl2]2 (150 mg,
0.24 mmol). Yield: 50%.
1H NMR (D2O, 500.10 MHz, 25 ꢀC):
d
¼ 1.22 (d, 3JH,H ¼ 7 Hz, 6H,
CH3cym-c), 2.30 (s, 3H, CH3), 2.42 (s, 3H, CH3cym-a), 2.62 (sept,
3JH,H ¼ 7 Hz, 1H, CHcym-b), 2.97 (br s, 4H, Hmor), 3.32e3.34 (m, 1H,
CH2eN), 3.86 (br s, 4H, Hmor), 3.97e3.99 (m, 1H, CH2eN), 5.97 (d,
3JH,H ¼ 7 Hz, 2H, Hcym), 6.15 (d, 3JH,H ¼ 7 Hz, 2H, Hcym), 7.54 (s, 1H,
H5) ppm. Elemental Anal. Calc for C21H28ClNO3OsS$0.75CH2Cl2: C,
39.35; H, 4.48; N, 2.10; Found: C, 39.16; H, 4.48; N, 2.39%. ESI-MSþ
m/z: 566.2 [M ꢁ Cl]þ.
1H NMR (CDCl3, 500.10 MHz, 25 ꢀC):
d
¼ 1.40e1.45 (m, 2H,
CH2pip), 1.56e1.63 (m, 4H, CH2pip), 1.70 (s, 15H, Cp*), 2.23 (s, 3H,
CH3), 2.55 (m, 4H, Hpip), 3.35 (d, 2JH,H ¼ 14 Hz, 1H, CH2eN), 4.03 (d,
2JH,H ¼ 14 Hz, 1H, CH2eN), 6.24 (s, 1H, H5) ppm. 13C NMR (CDCl3,
500.10 MHz, 25 ꢀC):
110.0 (C5), 91.3 (Cp*), 54.8 (NeCH3), 54.4 (CH2pip), 26.1 (CH2pip),
d
¼ 184.4 (C4), 163.5 (C6), 158.1 (C3), 150.4 (C2),
24.3 (CH2pip), 20.2 (C7), 9.0 (Cp*) ppm. Elemental Anal. Calc for
C
22H31ClNO3Rh: C, 53.29; H, 6.30; N, 2.82; Found: C, 53.46; H, 6.32;
Chlorido{3-(oxo-
k
O)-6-methyl-2-[(morpholin-4-yl)methyl]pyran-
h
N, 2.67%. ESI-MSþ m/z: 460.2 [M ꢁ Cl]þ.
4(1H)-thionato-
kS}(
5-1,2,3,4,5-pentamethylcyclopentadiene) rho-
dium(III) (6d).
Chlorido{3-(oxo-
k
O)-6-methyl-2-[(4-methylpiperazin-1-yl)methyl]
The reaction was performed analogous to the general
complexation procedure, using 3d (39 mg, 0.16 mmol), sodium
methoxide (9 mg, 0.16 mmol) and [Rh(Cp*)Cl2]2 (50 mg,
0.08 mmol). Yield: 55%.
pyran-4(1H)-onato-kO}(h
5-1,2,3,4,5-pentamethylcyclopentadienyl)
rhodium(III) (6c).
The reaction was performed analogous to the general
complexation procedure, using 3c (127 mg, 0.53 mmol), sodium
methoxide (29 mg, 0.53 mmol) and [Rh(Cp*)Cl2]2 (150 mg,
0.24 mmol). Yield: 51%.
1H NMR (MeOD, 500.10 MHz, 25 ꢀC):
d
¼ 1.73 (s, 15H, Cp*), 2.40
(s, 3H, CH3), 2.79 (br s, 4H, Hmor), 3.20e3.24 (m, 1H, CH2eN), 3.74
(br s, 4H, Hmor), 3.85e3.88 (m, 1H, CH2eN), 7.32 (s, 1H, H5) ppm.
Elemental Anal. Calc for C21H29ClNO3RhS$0.5CH2Cl2: C, 46.42; H,
5.44; N, 2.52; S, 5.76; Found: C, 46.49; H, 5.61; N, 2.40; S, 5.92%. ESI-
MSþ m/z: 467.3 [M ꢁ Cl]þ.
1H NMR (CDCl3, 500.10 MHz, 25 ꢀC):
d
¼ 1.70 (s, 15H, Cp*), 2.23
(s, 3H, CH3), 2.29 (s, 3H, NeCH3), 2.48 (br s, 4H, Hpaz), 2.67 (br s, 4H,
H
paz), 3.39 (d, 2JH,H ¼ 14 Hz, 1H, CH2eN), 4.12 (d, 2JH,H ¼ 14 Hz, 1H,
CH2eN), 6.24 (s, 1H, H5) ppm. 13C NMR (CDCl3, 127.75 MHz, 25 ꢀC):
d
¼ 185.5 (C4), 163.6 (C6), 158.3 (C3), 149.6 (C2), 110.0 (C5), 91.3
Single-crystal X-ray diffraction analysis
(Cp*), 55.0 (CH2paz), 53.9 (CH2paz), 52.8 (CH2eN), 46.2 (NeCH3paz),
20.2 (C7), 9.02 (Cp*) ppm. Elemental Anal. Calc for C22H32ClN2O3Rh
$ 0.5H2O: C, 50.83; H, 6.40; N, 5.39; Found: C, 51.08; H, 6.05; N,
5.24%. ESI-MSþ m/z: 474. [MꢁCl]þ, 375.4 [M-Cl-Mpip]þ.
Single crystals of 4a, suitable for X-ray diffraction analysis, were
obtained by the slow diffusion method from dichloromethane/n-
hexane and were measured on a X8 APEX2 diffractometer at 100 K.
A single crystal was positioned 40 mm from the detector and 914
frames with a 90 s exposure time over 1ꢀ scan width were
measured. SAINT was used to process the data and the structures
were solved by direct methods and refined by full-matrix least-
squares techniques [25]. Non-hydrogen atoms were refined with
anisotropic displacement parameters. Hydrogen atoms were
inserted at calculated positions and refined with a riding model.
The following programs for structure solution, refinement, molec-
ular diagrams and scattering factors were used: SHELXS-97 [26],
SHELXL-2013 [26], OLEX2 [27] SHELXLE [28] and ORTEP-3 [29].
Synthesis of the thiopyrone-based organometallic complexes
General procedure for the synthesis of thiopyrone-based organome-
tallic complexes.
Thiopyrone (1 eq) and sodium methoxide (1 eq) were dissolved
in dry methanol (10 mL, dried over molecular sieves 3 Å) and stirred
for 15 min under inert conditions. The solution was transferred into
a Schlenk tube containing the corresponding metal dimer (50 mg,
1 eq.) and the mixture was stirred for 1.5 h at room temperature.
The solvent was removed under reduced pressure, dissolved in
dichloromethane and filtered. The volume was reduced to ~2 mL,
the product precipitated upon addition of n-hexane and the
resulting suspension was stored at 4 ꢀC overnight. The formed
precipitate was removed by filtration, washed with n-hexane and
dried in vacuo.
Cytotoxicity in human cancer cell lines
Cell lines and culture conditions
Human CH1 (ovarian carcinoma) cells were kindly provided by
Lloyd R. Kelland (CRC Centre for Cancer Therapeutics, Institute of
Cancer Research, Sutton, UK) and SW480 (colon carcinoma) as well
as A549 (non-small cell lung cancer) cells by Brigitte Marian
(Institute of Cancer Research, Department of Medicine I, Medical
University of Vienna, Austria). Cells were grown in 75 cm2 culture
flasks (CytoOne, Starlab, Hamburg, Germany)) as adherent mono-
layer cultures in complete culture medium, i.e. minimal essential
medium (MEM) supplemented with 10% heat-inactivated fetal
Chlorido{3-(oxo-
4(1H)-thionato- S}(
The reaction was performed analogous to the general
complexation procedure, using 3d (39 mg, 0.16 mmol), sodium
methoxide (9 mg, 0.16 mmol) and [Ru(p-cym)Cl2]2 (50 mg,
0.08 mmol). Yield: 75%.
k
O)-6-methyl-2-[(morpholin-4-yl)methyl]pyran-
6-p-cymene)ruthenium(II) (4d).
k
h
1H NMR (D2O, 500.10 MHz, 25 ꢀC):
d
¼ 1.22 (d, 3JH,H ¼ 7 Hz, 6H,
CH3cym-c), 2.21 (s, 3H, CH3), 2.43 (s, 3H, CH3cym-1), 2.71 (sept,
3JH,H ¼ 7 Hz, 1H, CHcym-b), 2.99 (br s, 4H, Hmor), 3.32e3.34 (m, 1H,
CH2eN), 3.87 (br s, 4H, Hmor), 3.98e4.00 (m, 1H, CH2eN), 5.65 (d,
3JH,H ¼ 7 Hz, 2H, Hcym), 5.84 (d, 3JH,H ¼ 7 Hz, 2H, Hcym), 7.47 (s, 1H,
H5) ppm. Elemental Anal. Calc for C21H28ClNO3RuS$0.7CH2Cl2: C,
45.69; H, 5.19; N, 2.46; Found: C, 45.86; H, 5.43; N, 2.65%. ESI-MSþ
m/z: 476.3 [M ꢁ Cl]þ.
bovine serum, 1 mM sodium pyruvate, 4 mM L-glutamine and 1% v/
v non-essential amino acids (from 100ꢂ ready-to-use stock solu-
tion) (all purchased from SigmaeAldrich, Vienna, Austria). Cultures
were maintained at 37 ꢀC in a humidified atmosphere containing
95% air and 5% CO2.
Cytotoxicity tests in cancer cell lines
Cytotoxicity was determined by a colorimetric microculture
assay (MTT assay). For this purpose, CH1, A549, and SW480 cells
were harvested from culture flasks by trypsinization and seeded in
complete culture medium into 96-well microculture plates
(CytoOne, Starlab) in densities of 1 ꢂ 103 (CH1), 2 ꢂ 103 (SW480),
3 ꢂ 103 (A549) viable cells/well, respectively. After 24 h pre-
incubation, cells were exposed to dilutions of the test compounds
Chlorido{3-(oxo-
k
O)-6-methyl-2-[(morpholin-4-yl)methyl]pyran-
6-p-cymene)osmium(II) (5d).
4(1H)-thionato- S}(
k
h
The reaction was performed analogous to the general
complexation procedure, using 3d (31 mg, 0.16 mmol), sodium
methoxide (9 mg, 0.16 mmol) and [Os(p-cym)Cl2]2 (50 mg,
0.06 mmol). Yield: 79%.
Please cite this article in press as: M. Schmidlehner, et al., Journal of Organometallic Chemistry (2014), http://dx.doi.org/10.1016/
j.jorganchem.2014.10.044

M. Schmidlehner et al. / Journal of Organometallic Chemistry xxx (2014) 1e8
5
in 200
exposure, drug solutions were replaced with 100
mixture of RPMI 1640 medium (supplemented with 10% heat-
inactivated FBS and 4 mM -glutamine) and MTT solution (MTT
reagent in phosphate-buffered saline, 5 mg/mL). After incubation
for 4 h, medium was removed and the formazan product formed by
m
L/well complete culture medium for 96 h. At the end of
subsequent filtration, the products were obtained in moderate to
good yields by precipitation from dichloromethane/n-hexane
(Scheme 2, Table 1). We have recently shown that thiopyrone-
based organometallics provide an increased stability under physi-
ological conditions and promising anticancer potential in vitro. The
high affinity of sulfur to the ruthenium center prevents ligand
cleavage and inactivation of the complexes [18]. Therefore, 3d was
converted with the respective organometallic precursor yielding
4d, 5d and 6d. Small traces of water in the reaction solvent led to
decomposition of the formed complexes and formation of side
products during the conversion. It was essential to work strictly
under inert conditions. In addition, utilization of equimolar ratios of
the reactants reduces the amount of formed side products and fa-
cilitates the work up procedure. Overall, the complexes were iso-
lated in moderate to good yields (55e79%) and in high purity. All
synthesized compounds were characterized by 1D and 2D NMR
spectroscopy (Figs. S1e10), electrospray ionization mass spec-
trometry, elemental analysis and 4a by X-ray diffraction analysis. In
the case of the pyrone derivatives 3aec the shift of the H5 proton
was between 6.20 and 6.22 ppm, and the broad singlet for the
hydroxyl proton was at around 8.85 ppm. Formation of the Man-
nich products was confirmed by the disappearance of the H2 pro-
ton and the formation of new signals, assigned to the methylene
group between 3.45 and 3.49 ppm and the pendant cyclic amines
[30,31]. A downfield shift was observed for the H5 of the thionated
ligand 3d by around 1 ppm, which is typical for the thiopyrone
backbone [35]. The signal for the methyl group and the amino
moiety were found in the same area as for the O,O-analog.
mL/well of a 6:1
L
viable cells was dissolved in DMSO (150 mL/well). Optical densities
at 550 nm were measured with a microplate reader (Biotek
ELx808), using a reference wavelength of 690 nm to correct for
unspecific absorption. The percentage of viable cells was expressed
relative to untreated controls (defined as 100%), and 50% inhibitory
concentrations (IC50) were calculated from concentrationeeffect
curves by interpolation. Evaluation is based on at least three in-
dependent experiments (in case of IC50 > 200 mM on at least two
experiments), each comprising triplicates for each concentration
level.
Results and discussion
Thiopyrone-based organometallic complexes were found to be
highly active in human cancer cell lines; however, their solubility
under physiological conditions is significantly lower compared to
their pyrone analogs. This drawback can be prevented by the
insertion of highly hydrophilic groups such as morpholine, piperi-
dine or N-methylpyrazine. The modification of the pyrone scaffold
can be easily performed by Mannich reaction at position 6 of the
pyrone ring and has already been reported by Aytemir et al. [30,31]
Recently, Kasser et al. reported the synthesis of kojic acid-based
Mannich products and the corresponding ruthenium(II)-arene
complexes [19]. However, the organometallics were reported to
be insufficiently stable under physiological conditions, due to rapid
ligand cleavage. Direct thionation of the already reported ligands
was unsuccessful. Allomaltol was utilized as precursor for this
synthetic approach and was synthesized according to the protocol
via two-step synthesis. First, kojic acid was converted with thionyl
chloride to the respective chlorokojic acid and was subsequently
reduced with Zn/HCl to allomaltol [15,22,23]. Thioallomaltol 2 was
obtained by reaction of 1 with an equivalent amount of Lawesson's
reagent (Scheme 1) [32]. The utilization of Lawesson's reagent
compared to the reported synthesis with phosphorus pentasulfide
improved the yield to 80%, and the purification of the compound
can be easily performed via column chromatography [24,33].
The conversion to the respective Mannich products was ach-
ieved by the conversion of allomaltol 1 or thioallomaltol 2, with
equimolar amounts of 35% aqueous formaldehyde and the
respective cyclic secondary amine (morpholine, piperidine or N-
methylpiperazine) and refluxing the reaction mixture for 5 min.
After work up, the products were obtained in high yields (83e88%),
in the same range as the kojic acid analogs [19]. However, under
these reaction conditions, the yield of the already reported ligand
3b could be significantly increased from 30 to 88% [30].
The formation of the desired complexes was confirmed by disap-
pearanceof the eOHgroupatposition 3 and the shiftof therespective
arene protons upon coordination. Spectra of all O,O-chelating com-
plexes were recorded in CDCl3, due to the observed increased stability
of thecompoundsinthissolvent. Theprotonsoftheareneligandwere
observed asfourdoublets, and the two methyl groups alsosplit in two
doublets, which can be explained by the hindered inversion at the
metal center in aprotic solvents. Coordination of an organometallic
fragment to the (thio)pyrone ligand induced a diastereotopic splitting
into two doublets with a geminal coupling constant of 14 Hz. These
signals were unambiguously assigned to the methylene group in
position 2 by HSQC and HMBC experiments. A similar behavior was
observed for organometallic complexes bearing N-substituted 2-
pyridiones as chelating ligands [34]. This diastereotopic splitting of
the methylene group was observed for all synthesized metal com-
plexes. For the rhodium compounds 6aed the characteristic singlet
for the h
5-1,2,3,4,5-pentamethylcyclopentadienyl ligand (Cp*) was
found around 1.75 ppm.
The 1H NMR spectra of the thionated compounds of the d-series
were recorded in D2O, and methanol-d4 due to their solubility,
better resolution and insufficient stability in either CDCl3 or DMSO-
d6. Broadening of the aromatic signals of the p-cymene, which was
induced by coordination to the metal center, is obviously apparent
in the spectra of the osmium complex 5d. Compared to the arene
signals of the ruthenium analog 4d with 5.65 and 5.84 ppm there is
a downshift to 5.97 and 6.15 ppm. However, recording of 13C NMR
spectra was not possible for this series due to the surprising
The metal complexes were obtained according to established
literature procedures by deprotonation of the ligand with sodium
methoxide and reaction with the respective dimeric metal pre-
cursor [20,34]. After separation of byproducts by extraction and
Scheme 1. Synthesis of the (thio)pyrone precursors 1 and 2.
Please cite this article in press as: M. Schmidlehner, et al., Journal of Organometallic Chemistry (2014), http://dx.doi.org/10.1016/
j.jorganchem.2014.10.044

6
M. Schmidlehner et al. / Journal of Organometallic Chemistry xxx (2014) 1e8
Scheme 2. Synthesis of the O,O- and O,S-chelates and organometallic Ru(II) (4aed), Os(II) (5d) and Rh(III) (6aed) complexes.
insufficient stability and decomposition of the thiopyrone com-
plexes over the experiment time.
leads to a chiral center at the ruthenium atom and both enantiomers
were found in the unit cell in a 1:1 ratio.
Poor solubility can seriously affect the suitability as a drug [36].
For example, the maximum tolerated dose of KP1019 could not be
reached in the clinical setting due to solubility problems necessi-
tating large infusion volumes [37]. Overall, the attachment of cyclic
secondary amines such as piperazine, morpholine or piperidine
enhanced the solubility of the organometallic complexes tremen-
dously. The morpholine series showed the highest solubility of
240 mM for the ruthenium complex 4a and the rhodium analog 6a.
The solubility of 4b and 4c were found around 200 mM. The
rhodium complexes were soluble in the range of 60 mM for the
rhodium(III)-N-methylpiperazine derivative (6c). The complexes of
the d series were substantially less soluble, but even the osmium
analog (5d) showed 20 mM solubility.
Stability of ruthenium(II) complexes in aqueous solution
Most of the clinically applied anticancer agents are administered
intravenously, and therefore the stability in aqueous solution is a
crucial parameter for drug development. Those compounds should
act as prodrugs, which are activated by hydrolysis yielding the
highly reactive aqua-species [40,41]. The stability in water was
investigated by 1H NMR spectroscopy in D2O due to the high
aqueous solubility of the complexes. Samples were dissolved in D2O
and spectra were recorded over a period of 72 h. All synthesized
organometallics undergo hydrolysis to the corresponding aqua-
complexes within seconds. However, the thiopyrone-based com-
plexes were surprisingly very unstable, and even minor traces of
water induced decomposition, which was rather unexpected given
to the high stability reported for thiopyrone-based organo-
ruthenium complexes [15]. Even the osmium analogueanalog,
X-ray diffraction analysis
Single crystals of 4a, suitable for X-ray diffraction experiments,
were obtained by the slow diffusion method from dichloromethane/
n-hexane. The complex crystallized in the triclinic space group P-1
(Fig. 2). Details on the crystallographic measurements and the
crystal parameters are provided in Table 2. The complex adopts the
typical “piano-stool” configuration, where the deprotonated pyrone
acts as an anionic O,O-chelate forming a five-membered ring with an
envelope conformation. The RueO bond lengths 2.0819(19) Å
(RueO3) and 2.0877(20) Å (RueO2) were found to be in the same
range, which is in contrast to related structures [15,34,38,39]. This
behavior was only observed for the maltol-based complex described
by Peacock et al., where two solvent molecules formed hydrogen
bonds to the O,O-donor atoms [12]; ,however, no co-crystallized
solvent molecules were found in the case of 4a. The RueCl dis-
tance of 2.4162(8) Å is comparable to those of already published
Ru(II)-arene complexes [15]. The asymmetric coordination sphere
Table 1
Overview of synthesized complexes with corresponding yields.
Metal
Pyrone
Sec. amine
Yield
4a
4b
4c
4d
5d
6a
6b
6c
6d
Ru
Ru
Ru
Ru
Os
Rh
Rh
Rh
Rh
Allomaltol
Allomaltol
Allomaltol
Thioallomaltol
Thioallomaltol
Allomaltol
Allomaltol
Allomaltol
Morpholine
Piperidine
N-Methylpiperazine
Morpholine
Morpholine
Morpholine
Piperidine
N-Methylpiperazine
Morpholine
66%
73%
66%
75%
79%
64%
50%
51%
55%
Thioallomaltol
Fig. 2. Solid-state structure of complex 4a. Hydrogen atoms were omitted for clarity.
Please cite this article in press as: M. Schmidlehner, et al., Journal of Organometallic Chemistry (2014), http://dx.doi.org/10.1016/
j.jorganchem.2014.10.044

M. Schmidlehner et al. / Journal of Organometallic Chemistry xxx (2014) 1e8
7
Table 2
which should be the most stable of this series according to the
HSAB principle, was far too unstable under the applied conditions
(Figs. S12 and 13).
Crystallographic data, data collection parameter und structure refinement de-
tails for 4a.
4a
Interestingly, only complex 4a of the ruthenium series showed
sufficient stability attributed to the crucial influence of the N-het-
erocycle of the synthesized organometallic compounds. In Fig. 3,
the aromatic region of 4a is illustrated, and the formation of
dimeric species, [Ru(p-cym)2(OH)3]þ, can be observed over time
(see Fig. S11 for complete spectra). After 3 days less than 10% of
complex 4a reacted to the dimeric species; however, complex 4c
was significantly less stable, where the same amount of dimer was
formed after 6 h. With piperidine as secondary amine, after 5 min
around 7% and after 6 h over 20% of the ruthenium complex 4b was
converted to the dimeric species. NMR experiments confirmed that
the rhodium complexes 6aec were stable in aqueous solution for
over 72 h without any traces of ligand cleavage or decomposition
and are therefore suitable for biological testing.
Empirical formula
Formula weight [g/mol]
Temperature/K
Wavelength/Å
Crystal size/mm
Crystal system
Space group
a [Ǻ]
C21H28ClNO4Ru
494.97
100(2)
0.71073
0.08 ꢂ 0.08 ꢂ 0.1
triclinic
P-1
9.2542(4)
10.3922(4)
12.7027(5)
67.543(2)
70.582(3)
68.259(2)
1022.41(7)
2
1.608
0.925
508
1.78e27.49ꢀ
ꢁ12 ꢃ h ꢃ 11
ꢁ13 ꢃ k ꢃ 13
ꢁ16 ꢃ l ꢃ 16
17,207/4609
4609/15/222
0.0300
b [Ǻ]
c [Ǻ]
a
b
g
[ꢀ]
[ꢀ]
[ꢀ]
Volume [Ǻ3]
Ζ
Calculated density/mg/m3
Absorption coefficient/mmꢁ1
F(000)
Due to the obtained stability data, 4bed, 5d and 6d were not
further investigated in biological experiments, and only com-
pounds 4a and the Rh(III) complexes 6aec were tested for their
cytotoxicity.
Q
range for data collection
Index ranges
Reflections collected/unique
Data/restraints/parameters
R(Int)
Cytotoxicity
The investigated complexes 4a and 6aec were tested for their
cytotoxic activity in three human cancer cell lines, namely CH1
(ovarian carcinoma), SW480 (colon carcinoma), and A549 (non-
small cell lung cancer), by means of the colorimetric MTT assay
(Table 3, Fig. S14). In the rather chemoresistant cell line A549, IC50
Final R indices [I > 2
s
(I)]
a
R1
0.0343
0.0853
1.022
b
wR2
GOFc
a
R1 ¼ SjjFoj ꢁ jFcjj/SjFoj.
b
c
wR2 ¼ {S[w(F2o ꢁ F2c)2]/S[w(Fo2)2]}1/2
.
values greater than 200
also holds true for SW480 cells, with the only exception being the
Rh-piperidine derivative 6b, which yielded an IC50 of about 100 M.
mM were observed for all complexes. This
GOF ¼ {S[w(F2o ꢁ Fc2)2]/(n ꢁ p)}1/2, where n is the number of reflections and p
is the total number of parameters refined.
m
Fig. 3. 1H NMR spectra of 4a in D2O after a) 5 min, b) 3 h, c) 24 h, d) 48 h and e) 72 h showing the formation of [Ru2(p-cym)2(OH)3]þ at 5.1 and 5.4 ppm, respectively.
Please cite this article in press as: M. Schmidlehner, et al., Journal of Organometallic Chemistry (2014), http://dx.doi.org/10.1016/
j.jorganchem.2014.10.044

8
M. Schmidlehner et al. / Journal of Organometallic Chemistry xxx (2014) 1e8
Table 3
The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.
uk/data_request/cif.
Solubility and cytotoxicity of complexes (4a, 6aec) in three human cancer cell lines
in comparison with the Rh-allomaltol complex and NKP-1339 [42,43].
Compound
Solubility [mM]
IC50 [m
M]a
Appendix B. Supplementary data
A549
CH1
56
199 42
136 27
201 29
104 20
SW480
4a
6a
6b
6c
240
240
100
70
35
18
>200
>200
>200
>200
>200
5
>200
>200
109 29
>200
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.jorganchem.2014.10.044.
Rh-allomaltol
NKP-1339
145
5
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62
9
88 19
a
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50% inhibitory concentrations in A549, CH1 and SW480 cells in the MTT assay
standard deviations obtained from two
(96 h exposure). Values are means
(if > 200) or three independent experiments.
In the cell line CH1, as the most chemosensitive one, all complexes
showed activity in the tested concentration range. In this cell line,
the Ru complex 4a exhibited an IC50 value of about 60 mM, which is
more potent than the Rh analog 6a and in the range of cytotoxicity
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Acknowledgments
We thank the University of Vienna for financial support.
Appendix A. Supplementary material
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€
€ €
[42] O. Domotor, S. Aicher, M. Schmidlehner, M.S. Novak, A. Roller, M.A. Jakupec,
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W. Kandioller, C.G. Hartinger, B.K. Keppler, E.A. Enyedy, J. Inorg. Biochem. 134
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CCDC 1007969 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge from
[43] P.-S. Kuhn, V. Pichler, A. Roller, M.A. Jakupec, W. Kandioller, B. Keppler, sub-
mitted for publication.
Please cite this article in press as: M. Schmidlehner, et al., Journal of Organometallic Chemistry (2014), http://dx.doi.org/10.1016/
j.jorganchem.2014.10.044