Synthesis and biological activities of transition metal complexes based on acetylsalicylic acid as neo-anticancer agents

Article abstract of DOI:10.1021/jm101019j

[(μ⁴-ν²)-(Prop-2-ynyl)-2-acetoxybenzoate] dicobalthexacarbonyl (Co-ASS), a derivative of aspirin (ASS), demonstrated high growth-inhibitory potential against various tumor cells with interference in the arachidonic acid cascade as probable mode of action. The significance of the kind of metal and cluster was verified in this structure-activity study: Co ₂(CO)₆ was respectively exchanged by a tetrameric cobalt-, trimeric ruthenium-, or trimeric ironcarbonyl cluster. Furthermore, the metal binding motif was changed from alkyne to 1,3-butadiene. Compounds were evaluated for growth inhibition, antiproliferative effects, and apoptosis induction in breast (MCF-7, MDA-MB 231) and colon cancer (HT-29) cell lines and for COX-1/2 inhibitory effects at isolated isoenzymes. Additionally, the major COX metabolite prostaglandin E2 (PGE₂) was quantified in arachidonic acid-stimulated MDA-MB 231 breast tumor cells. It was demonstrated that the metal cluster was of minor importance for effects on cellular activity if an alkyne was used as ligand. Generally, no correlation existed between growth inhibition and COX activity. Cellular growth inhibition and antiproliferative activity at higher concentrations of the most active compounds Prop-ASS-Co ₄ and Prop-ASS-Ru₃ correlated well with apoptosis induction.

Full text of DOI:10.1021/jm101019j

J. Med. Chem. 2010, 53, 6889–6898 6889
DOI: 10.1021/jm101019j
Synthesis and Biological Activities of Transition Metal Complexes Based on Acetylsalicylic Acid
as Neo-Anticancer Agents
Gerhard Rubner,† Kerstin Bensdorf,† Anja Wellner,† Brigitte Kircher,‡ Silke Bergemann,† Ingo Ott,§ and Ronald Gust*,†,
†
‡
€
€
Institute of Pharmacy, Freie Universitat Berlin, Konigin-Luise-Strasse 2 þ 4, 14195 Berlin, Germany, Immunobiology and
Stem Cell Laboratory, Innsbruck Medical University, Department of Internal Medicine V;Hematology & Oncology, Anichstrasse 35,
A-6020 Innsbruck, Austria, §Institute of Pharmacy, TU Braunschweig, Beethovenstrasse 55, 38106 Braunschweig, Germany, and
Department of Pharmaceutical Chemistry, Institute of Pharmacy, University of Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria
Received February 19, 2010
[(μ4-η2)-(Prop-2-ynyl)-2-acetoxybenzoate]dicobalthexacarbonyl (Co-ASS), a derivative of aspirin (ASS),
demonstrated high growth-inhibitory potential against various tumor cells with interference in the
arachidonic acid cascade as probable mode of action. The significance of the kind of metal and cluster
was verified in this structure-activity study: Co2(CO)6 was respectively exchanged by a tetrameric cobalt-,
trimeric ruthenium-, or trimeric ironcarbonyl cluster. Furthermore, the metal binding motif was changed
from alkyne to 1,3-butadiene. Compounds were evaluated for growth inhibition, antiproliferative effects,
and apoptosis induction in breast (MCF-7, MDA-MB 231) and colon cancer (HT-29) cell lines and for
COX-1/2 inhibitory effects at isolated isoenzymes. Additionally, the major COX metabolite prostaglandin
E2 (PGE2) was quantified in arachidonic acid-stimulated MDA-MB 231 breast tumor cells. It was
demonstrated that the metal cluster was of minor importance for effects on cellular activity if an alkyne
was used as ligand. Generally, no correlation existed between growth inhibition and COX activity. Cellular
growth inhibition and antiproliferative activity at higher concentrations of the most active compounds Prop-
ASS-Co4 and Prop-ASS-Ru3 correlated well with apoptosis induction.
Introduction
the significance of the [alkyne]dicobalthexacarbonyl cluster
for antitumor activity.
Platinum-based drugs such as cisplatin or oxaliplatin are
widely used in the treatment of cancer although they induce
strong side effects (e.g., nausea or alopecia) and develop
resistance during therapy.1 Therefore, many groups searched
for nonplatinum complexes with another mode of action
usable in second-line therapy.2
In our group, we intensively investigated Co-ASS (formula
see Scheme 2), a derivative of aspirin (ASS).3-8 We assumed a
selective interference in the arachidonic acid cascade leading
to a growth inhibition of cyclooxygenase (COXa) dependent
tumor cells. Indeed, this organometallic drug was an effective
COX inhibitor andshowedstrong growth-inhibitoryeffectsat
mammary, colon, and leukemia cell lines.3,4 For the induction
of strong effects, however, the ligand has to be a derivative
of aspirin. The exchange of the 2-acetoxybenzoyl substructure
in Co-ASS by other nonsteroidal anti-inflammatory drugs
(NSAID) strongly reduced the activity.4 In continuation of
this structure-activity relationship study, we tried to consider
The mode of action of Co-ASS is not completely resolved
but might involve the inhibition of the COX enzymes prob-
ably by CO poisoning the haem iron center.9 Therefore, the
dicobalthexacarbonyl moiety was exchanged in a first step by
a higher cobalt- (Co4(CO)10)10 and related ruthenium- and
ironcarbonyl cluster (Ru3(CO)9;Fe3(CO)9) toincreasetheCO
proportion compared to the ASS ligand.11,12
In the next step, the influence of the alkyne bonding was
studied on the example of the Ru3(CO)9 and Fe3(CO)9 com-
plexes. For this purpose, the alkyne was exchanged by an
1,3-butadiene moiety. Further modifications were the use of
but-2-yne-1,4-diyl-bis(2-acetoxybenzoate) (Di-ASS) as ligand
bearing two ASS and the use of ferrocene as metal unit.
For comparison with Co-ASS, all ligands and complexes
were tested for growth inhibition of MCF-7, MDA-MB 231,
and HT-29 cells for COX inhibitory effects at isolated enzymes
(COX-1 and COX-2) as well as arachidonic acid-stimulated
breast cancer cells (MDA-MB 231) using the intracellular
content of the major metabolite prostaglandin E2 as param-
eter. Antiproliferative and proapoptotic effects were studied
with the most impressive compounds.
*To whom correspondence should be addressed. Phone: þ43 512 507 5245.
Fax: þ43 512 507 2940. E-mail: ronald.gust@uibk.ac.at. Address:
Department of Pharmaceutical Chemistry, Institute of Pharmacy,
University of Innsbruck, Innrain 52a, A-6020 Innsbruck, Austria.
a Abbreviations: COX, cyclooxygenase; NSAID, nonsteroidal anti-
Inflammatory drug; HR-MS, high resolution mass spectrometry; FAB-MS,
fast atomic bombardment mass spectrometry; EI-MS, electron ioniza-
tion mass spectrometry; ESI-TOF MS, electrospray ionization time-of-
flight mass spectrometry; TLC, thin layer chromatography; ELISA,
enzyme-linked immunoabsorbant assay; PGE2 prostaglandin E2;
DMEM, Dulbecco’s Modified Eagle Medium; OD, optical density;
ssDNA, single-stranded DNA; PBS, phosphate-buffered saline.
Results and Discussion
Synthesis and Molecular Characterization. The reaction of
alkynoles or alkadienoles and 2-aetoxybenzoyl chloride13
(ASSCl) at 0 °C gave the respective ester. Pyridine as base
bound liberated HCl.
r
2010 American Chemical Society
Published on Web 09/21/2010
pubs.acs.org/jmc

6890 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Scheme 1. Synthesis of Ligands
Rubner et al.
Penta-2,4-dien-1-ol, which was not commercially avail-
able, was synthesized as E/Z mixture from bromoethyl-
acetate and acroleine via Wittig reaction14 and subsequent
reduction15 with LiAlH4. Esterification with ASSCl yielded
pentadiene-ASS.
All metal carbonyl syntheses were carried out under Schlenk
conditions using an inert, dry atmosphere. The progress of the
reactions could be followed by CO release (Scheme 1).
Co4(CO)12 was freshly prepared by heating an n-hexane
solution of Co2(CO)8 for 5 h to reflux.16 After addition of the
appropriate ligand, the dark-blue complexes were formed at
room temperature within three days with yields from 48% to
69% (Scheme 2). The building of Co-ASS was avoided by
using Co4(CO)12 and the ligands in a molar ratio of 1:1 and a
reaction temperature not higher than room temperature.10
Degradation or side products were separated by column
chromatography on silica gel.
Tetracobaltdecacarbonyl complexes can be best charac-
terized by IR spectroscopy.10,16 Gervasio et al. solved the
structure of [alkyne]tetracobaltdecacarbonyl in 1984 and
demonstrated the direct linkage of the alkyne to two neigh-
boring Co(CO)2 centers. Each Co atom build further bonds
to the metals of a Co2(CO)6 unit with four terminal and two
bridged CO ligands. In accordance with this structure, CO
bands appeared in the spectra at 1750-1880 cm-1 (bridged
CO) and about 2000-2100 cm-1 (terminal CO).17
In the IR spectrum of Prop-ASS-Co4, the characteristic
CtC-H vibrations at 3240 (ν(C-H)) and 2126 cm-1
(ν(CtC)) of the unbound ligand are replaced by ethylene-
like absorptions at 2958 and 1628 cm-1 in the complex. The
carbonyl bands appear at 1869 cm-1 (bridged) as well as
2033, 2055, and 2097 cm-1 (terminal).
In the 13C NMR spectra of the cobalt complexes, the
terminal and bridged carbonyls showed resonances at about
200 ppm, comparable to those described by Lang et al.18 The
O-CH2 group of Prop-ASS is shifted from 52.73 to 65.5 ppm
and the alkyne carbons from 77.52 and 75.44 to 72.2 and
74.9 ppm, respectively. The NMR resonances allow the
distinction of Prop-ASS-Co4 from Co-ASS (1H NMR:
δ (-O-CH2-)=5.47; 13C NMR: δ (-CtC-H))d 72.88 and
88.42) and excluded a contamination of Prop-ASS-Co4 with
its dicobalthexacarbonyl derivative.
Whereas the formation of [alkyne]tetracobaltdecacarbonyl
cluster was accessible at room temperature, Ru3(CO)12 and
the respective ligands had to be refluxed in n-hexane under
argon atmosphere for at least 6 h to obtain the trinuclear
ruthenium complexes. The separation of Prop-ASS-Ru3 and
But-ASS-Ru3 from their reaction mixtures (yields from 6%
to 17%) was successful using column chromatography on
silica gel.
Their structural assignment to [alkyne]trirutheniumnona-
carbonyl complexes based on publications from 1970s19,20
but also on actual studies (e.g., on dicobalt/triruthenium
carbonyl clusters21). These papers describe the crystal struc-
tures of complexes, which consist of a triangular array of
ruthenium atoms with an alkyne ligand bound to one face
of the cluster in a μ3,η2-coordination mode and with the
carbon-carbon bond positioned perpendicular to one Ru-
Ru edge. At each metal, three CO are terminally bound.
In accordance with this structure, the IR spectra exhibited
carbonyl bands in the range of 1960-2095 cm-1. No band of
a bridged CO near 1878 cm-1 as observed in the Ru3(CO)10
cluster was present.22,23 The latter can be obtained using
activated ruthenium cluster (e.g., Ru3(CO)10(MeCN)2).23
In accordance with a Ru3(CO)9 structure, the IR band
of Prop-ASS-Ru3 and But-ASS-Ru3 showed bands at
2005-2080 cm-1 without a resonance of bridged CO. The
1H NMR spectra of the ruthenium complexes followed the
trend of the cobalt complexes. The methylene group in
e.g. Prop-ASS-Ru3 is shifted from 4.88 ppm to 5.47 ppm
and the alkyne proton from 2.53 to 6.11 ppm. The resonance
Because of the magnetic properties of the cobalt cluster,
NMR spectra can only be used for structural characteriza-
tion with restrictions. The 1H NMR signals of the alkynol or
the alkyndiol moiety are broadened, but characteristically
shifted to lower field upon complexation (e.g., Prop-ASS-
Co4: -O-CH2- from δ = 4.88 to δ = 5.03; tC-H from δ =
2.53 to δ = 6.10).

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6891
Scheme 2. Synthesis of Alkyne Complexes
of the methyl group in But-ASS-Ru3 is not as strongly
influenced and appeared at 2.66 ppm.
nor the target compound were detected by thin layer chro-
matography (TLC).
These spectra pointed to the presence of a nonhydrido
triruthenium clusters (Prop-ASS-Ru3: one proton singlet
resonance at 6.11 ppm). It is well-known that the heating
of Ru3(CO)12 with alkynes giving oxidative addition of the
terminal or an internal hydrogen to the Ru cluster.22 There-
fore, the building of the hydrido-alkynyl complexes Prop-
ASS-Ru3H and But-ASS-Ru3H or the hydrido-allyl complex
allyl-But-ASS-Ru3H during the synthesis of Prop-ASS-Ru3
and But-ASS-Ru3 cannot be excluded (separation as side
products by column chromatography is very likely).
The best route for the synthesis of [alkyne]triironnona-
carbonyl complexes was the reaction of Fe3(CO)12 and the
appropriate alkyne in low boiling solvents (Scheme 2). How-
ever, from the title compounds only But-ASS-Fe3 could
be obtained in small amounts, although trimethylamine-
N-oxide as catalyst already increased the yields.25 Further-
more, the attempt failed to optimize the synthesis by varia-
tion of the solvent. Only n-pentane offered satisfying yields.
Nevertheless, it was impossible to get Prop-ASS-Fe3 and
Di-ASS-Fe3.
If the alkyne in Prop-ASS is replaced by a diene moiety
(pentadiene-ASS), the coordination to Ru3(CO)12 is possible
by a η4-bond.19 Besides a trinuclear complex, the presence of
its mononuclear similarity pentadiene-ASS-Ru is possible.24
Pentadiene-ASS-Ru3 (Scheme 3) was separated from the
reaction mixture by column chromatography. The light-yellow
powder which was obtained in low yield (8%) was character-
ized by IR spectroscopy and high resolution mass spectrometry
(HR-MS). The two characteristic bands at 2034 and 1948 cm-1
in the IR spectra indicated terminal CO bands but did not allow
a differentiation from pentadiene-ASS-Ru. However, the MS
studies (FAB- and EI-MS) clearly point to a trinuclear com-
plex. Unfortunately, attempts failed to gain structural infor-
mation by NMR spectroscopy, so a possible rearrangement
building a hydrido complex22 cannot be excluded.
Interestingly, in the case of But-ASS-Fe3, a further orange
colored complex (Di(But-ASS)-Fe3) was isolated from the
reaction mixture. MS spectra clearly indicated the presence
of two But-ASS ligands at a triironoctacarbonyl cluster.
Triironnonacarbonyl clusters with one alkyne ligand are
well-known in the literature.25 We propose a comparable
structure of But-ASS-Fe3 in which the acetylene moiety of
the ligand is perpendicularly oriented over the Fe3 triangle.
In accordance with this structure, the IR spectra exhibited
ν(CO) bands at 2061, 2026, and 1986 cm-1
.
[η4-Alkadienyl]irontricarbonyl complexes were accessible
via two synthetic routes: They can be obtained by UV
irradiation of an n-hexane solution of the ligand and Fe(CO)5
in a cooled quartz glass tube26 or by refluxing the ligand
together with Fe2(CO)9 in diethyl ether.27 Fe2(CO)9 was
previously produced in glacial acetic acid from Fe(CO)5 by
irradiation with UV light.28 It turned out that the second
It should be noted that it was impossible to coordinate
hexadiene-ASS to Ru3(CO)12. Neither decomposition products

6892 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Scheme 3. Synthesis of Alkadiene and Ferrocene Complexes
Rubner et al.
Table 1. Growth-Inhibitory Effects (IC50 Values [μM]) on Breast
Cancer and Colon Carcinoma Cell Lines
route led to much higher yields and was therefore preferably
used.
The Fe coordination can be confirmed by 1H NMR
spectroscopy.29,30 The O-CH2 group in hexadiene-ASS is
high field shifted from 5.78 to 4.26 ppm, the methyl group
from 1.79 to 1.43 ppm. The most characteristic effects
occurred at C2 and C5, with a shift of the isochronic protons
from 4.79 ppm to 1.30 ppm (C5-H) and 1.08 ppm (C2-H),
and in a much lower extent at C3-H (6.08 to 5.08 ppm) and
C4-H (6.29 to 5.27 ppm).
compd
cisplatin
MCF-7
MDA-MB 231
HT-29
2.0 ( 0.3
1.4 ( 0.3
>50
3.3 ( 0.5
1.9 ( 0.3
>50
2.4 ( 0.4
9.8 ( 3.0
>50
Co-ASS
aspirin
Prop-ASS-Co4
But-ASS-Co4
Di-ASS-Co4
1.9 ( 0.2
11.4 ( 0.4
4.7 ( 0.2
3.5 ( 0.2
15.0 ( 1.5
2.1 ( 1.3
2.7 ( 0.7
11.1 ( 0.0
8.0 ( 1.3
As already described above, pentadiene-ASS existed as an
E/Z-mixture and was reacted with Fe(CO)5. From the reac-
tion mixture only the complex of the E-isomer was isolated as
indicated by the NMR resonances, which were very similar
to those of hexadiene-ASS-Fe from which the E-isomer was
used for coordination.
The IR spectra showed two characteristic bands at about
1970 and 2050 cm-1 of [η4-s-cis-1,3-butadiene]Fe(CO)3.26 In
the case of the [η4-s-trans-1,3-butadiene]Fe(CO)3 isomer,
these vibrations would appear at about 30 cm-1 lower
frequencies.26
To study the relevance of the carbonyl cluster for bio-
logical effects, we combined ASS with a ferrocene moiety
by reaction of 2-acetoxybenzoyl chloride with ferrocenyl
carbinol (ferrocene-ASS) (Scheme 3). Ferrocenyl carbinol
can either be produced from ferrocene with formaldehyde
and sulphuric acid or by reduction of the commercially
available ferrocene carboxaldehyde with LiAlH4.31
In Vitro Chemosensitivity Assay. Antitumor activity was
determined in vitro using mammary carcinoma (MCF-7 and
MDA-MB 231) as well as colon (HT-29) cell lines. Cisplatin
and Co-ASS were used as reference.
Prop-ASS-Ru3
But-ASS-Ru3
Di-ASS-Ru3
1.4 ( 0.2
4.1 ( 0.6
5.5 ( 0.5
2.4 ( 0.0
17.8 ( 0.0
7.6 ( 0.7
2.2 ( 0.2
6.4 ( 0.0
10.1 ( 0.1
pentadiene-ASS-Ru3
10.0 ( 1.7
7.5 ( 0.4
19.8 ( 1.9
pentadiene-ASS-Fe
hexadiene-ASS-Fe
But-ASS-Fe3
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
>20
ferrocene-ASS
slightly more active against MDA-MB 231 cells (IC50 = 1.9
and 3.3 μM, respectively) but less active at the HT-29 cell line
(IC50= 9.8 and 2.4 μM, respectively; Table 1).
All ligands as well as aspirin, Co4(CO)12, CoCl2, Fe(CO)5,
FeCl2, Ru3(CO)12, and RuCl3 were separately tested up to
concentrations of 20-50 μM and demonstrated no inhibi-
tory effects (data not shown).
Prop-ASS-Co4 and Co-ASS induced comparable growth
inhibition at the breast cancer cell lines. The stability of the
Co4(CO)10 cluster in solution was already described,10 so a
break down into Co-ASS can be excluded. This assumption
confirmed the results with HT-29 cells at which Prop-
ASS-Co4 was distinctly more active than Co-ASS and even
reached the IC50 values of cisplatin (Table 1).
Elongation of the spacer length between the [alkyne]cobalt
cluster and the ASS moiety drastically reduced the cellular
growth. But-ASS-Co4 possessed IC50 values at all cell lines
higher than 10 μM. The related dicobalthexacarbonyl compounds
Because all compounds showed their maximum of activity
in a time-dependent growth-inhibition test after 72-96 h
(data not shown), the IC50 value was determined after an
incubation time of 72 h.
Co-ASS5 caused comparable effects to cisplatin against
MCF-7 cells (IC50 = 1.4 and 2.0 μM, respectively) and was

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6893
Table 2. Antiproliferative Activity of Prop-ASS-Ru3 and Prop-ASS-Co4
against MDA-MB 231 Cells
Table 3. COX-Inhibitory Effects and PGE2 Inhibition at a Concentration
of 10 μM
cell proliferation after
an incubation time of
compd
COX-1 (%)
COX-2 (%)
PGE2 (%)
aspirin
Co-ASS
29.2 ( 2.0
68.0 ( 5.4
1.0 ( 0.1
63.0 ( 5.0
0
compd
control
conc (μM)
24 h (%)
100
48 h (%)
100
72 h (%)
100
40.0 ( 5.0
Prop-ASS-Co4
But-ASS-Co4
Di-ASS-Co4
38.8 ( 1.9
20.3 ( 1.9
22.0 ( 4.3
39.9 ( 1.6
22.0 ( 1.2
22.4 ( 5.5
6.7 ( 0.1
5.1 ( 0.3
0
Prop-ASS-Ru3
Prop-ASS-Ru3
5
10
52.9 ( 8.1 41.9 ( 17.7 39.0 ( 29.0
17.2 ( 1.9 0.6 ( 0.2 0.4 ( 0.3
Prop-ASS-Co4
Prop-ASS-Co4
5
10
56.1 ( 5.3 57.1 ( 13.2 44.3 ( 2.5
37.7 ( 5.9 24.2 ( 39.2 0.9 ( 0.6
Prop-ASS-Ru3
But-ASS-Ru3
Di-ASS-Ru3
42.7 ( 0.9
35.1 ( 1.1
67.3 ( 9.4
33.0 ( 1.2
13.8 ( 2.3
23.6 ( 1.8
a
0
29.5 ( 1.8
were nearly inactive (IC50 values of But-ASS-Co2 and
Di-ASS-Co2 > 20 μM).
pentadiene-ASS-Ru3
87.7 ( 1.6
56.3 ( 10.7
a
A second ASS at the Prop-ASS ligand (Di-ASS-Co4) did
not increase the cell growth-inhibitory capacity. Di-ASS-Co4
was comparably active as Prop-ASS-Co4 at MDA-MB 231
cells but was less active against MCF-7 (IC50 = 4.7 μM) and
HT 29 cells (IC50 = 8.0 μM).
The exchange of the Co4(CO)10 cluster in Prop-ASS-Co4
by Ru3(CO)9 (Prop-ASS-Ru3) did not change the cell
growth-inhibitory properties. For both compounds, nearly
identical IC50 values were calculated (Table 1). If the same
modification was done at But-ASS-Co4, the growth inhibi-
tion of MCF-7 and HT-29 cells (IC50 ≈ 5 μM) increased.
Interestingly, in the case of Di-ASS-Ru3, the activity at the
MDA-MB 231 cell line was reduced (IC50 =7.6 μM) com-
pared to Di-ASS-Co4 (IC50 = 2.1 μM). The growth inhibi-
tion of MCF-7 and MDA-MB 231 cells, however, remained
mainly unchanged.
From the data listed in Table 1, it is obvious that an alkyne
ligand seems to be essential for high growth-inhibitory
activity. Pentadiene-ASS-Ru3 reduced the growth of breast
cancer cells only marginally and was nearly inactive at
HT-29 cells. The related iron complexes pentadiene-ASS-Fe
and hexadiene-ASS-Fe and ferrocene-ASS were completely
inactive.
Generally, the ruthenium and cobalt complexes induced
stronger effects than aspirin. Therefore, their effect on the
proliferation of MDA-MB 231 and MCF-7 cells was studied
on the example of Prop-ASS-Ru3 and Prop-ASS-Co4 by thymi-
dine uptake. As shown in Table 2, both compounds time-
dependently inhibited the proliferative activity of MDA-MB
231 cells. After a 48 h incubation, 10 μM of Prop-ASS-Ru3 even
completely blocked proliferation. Similar results were observed
with MCF-7 cells (data not shown).
COX and PGE2 Assay. Aspirin belongs to the class of
NSAIDs, whose pharmacological effects are based on their
ability to inhibit cyclooxygenase enzymes. NSAIDs have
also attracted attention as novel cytostatics, as clinical studies
proved positive therapeutic effects for cancer patients.32
Furthermore, it was found that the COX-2 isoenzyme is over-
expressed in various tumors, and elevated levels of the products
of cyclooxygenase (prostaglandins) were detected.
Therefore, we studied the influence of the organometallic
compounds for their COX-1 and COX-2 inhibiting profile
and quantified the levels of the major COX metabolite
prostaglandin E2 (PGE2) in arachidonic acid-stimulated
MDA-MB 231 breast tumor cells by enzyme-linked immuno-
absorbant assay (ELISA) (Table 3).
pentadiene-ASS-Fe
hexadiene-ASS-Fe
But-ASS-Fe
33.7 ( 3.3
16.1 ( 1.3
11.5 ( 1.3
25.5 ( 1.3
5.2 ( 0.4
0
28.5 ( 1.7
24.6 ( 4.2
21.9 ( 1.5
0
2.5 ( 0.1
0
ferrocene-ASS
a Not evaluable because of too high growth-inhibitory effects.
both isoenzymes approximately to the same extent.4 Modifi-
cation at the ligand reduced the COX-inhibitory effects as
well as the cellular growth-inhibiting capacity.4 Therefore,
it was assumed that interference in the arachidonic acid
cascade is part of the mode of action.
To confirm this hypothesis, the COX-1 and COX-2 in-
hibitory potential of all organometallic compounds synthe-
sized in this study was determined by ELISA at isolated
enzymes and in a cellular system using MDA-MB 231 cells
(COX-1) by PGE2 assay.
At 10 μM, Co-ASS inhibited both cyclooxygenase
enzymes to 68% (COX-1) and 63% (COX-2), respectively.
Therefore, we used this concentration and performed the
ELISA without calculation of the IC50 values.
Aspirin was only marginally active at COX-1 (29% in-
hibition) and inactive at COX-2. The missing activity of all
ligands at both isoenzymes documented the relevance of the
metal cluster for COX-inhibitory effects.
The kind of cluster is also of greater importance than the
number of CO ligands. The [alkyne]Co4(CO)10 or [alkyne]-
Ru3(CO)9 complexes mediated lower COX inhibition than
Co-ASS. Only Di-ASS-Ru3 reached at COX-1 the same
efficacy (67.3%) as Co-ASS.
It might be possible that the size of the higher cluster
hindered the attachment of the drug to the active site so
poisoning of the haem inside the enzyme did not occur.
Nevertheless, acetylation of amino acids at the entrance
channel comparable to Co-ASS (Lys 346) might be
possible33 but was not yet investigated in detail.
Pentadiene-ASS-Ru3 was identified as most COX-active
compound among the new complexes. It was comparably
active at COX-2 and even more active then Co-ASS at COX-1
(87.7% at 10 μM). Exchange of the Ru3(CO)9 cluster by
Fe(CO)3 drastically reduced the inhibitory effects (see
Table 3). The same is true if ferrocene (ferrocene-ASS) is
used as metal moiety.
The investigations on the PGE2 level in MDA-MB 231
cells, however, has to be interpreted with caution because the
compounds reduced the cell number at a concentration of
10 μM and in some cases even no cellular growth was detected
after 24 h anymore. Only Di-ASS-Ru3, pentadiene-ASS-Fe,
and hexadiene-ASS-Fe suppressed the PGE2 level by about
20-30%.
As already described, Co-ASS inhibited isolated COX-1
and COX-2 more efficiently than the parent compound
aspirin.4 The preferential inhibition of COX-1 by aspirin
was not observed with the metal complex, which inhibited

6894 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Rubner et al.
Figure 1. Caspase-3 activity compared to untreated control (drug concentration 10 μM) of selected compounds.
Figure 2. Time-dependent apoptosis induction by Prop-ASS-Ru3 and Prop-ASS Co4. Apoptosis in the absence of the compounds was set at 1.
Antiapoptotic Activity. Since it is well-known that COX
inhibition can induce cell cycle arrest and apoptosis,34 we
studied apoptosis induction by activation of caspase-3 in
MDA-MB 231 and by single-stranded DNA (ssDNA) for-
mation as well as acridine orange staining in MDA-MB 231
and MCF-7 cells.
The reference Co-ASS strongly activated programmed cell
death indicated by a 5.19 times higher caspase-3 level after a
24 h incubation compared to untreated cells.8 This effect
depended on the metal cluster. The Co4(CO)10 and Ru3(CO)9
analogues were less effective. Interestingly, Prop-ASS-Ru3
showed medium effectiveness compared to Co-ASS and
Prop-ASS-Co4 (see Figure 1). The attachment of a second
ASS moiety at the Prop-ASS did not increase the apoptosis
induction of the Co and Ru complexes.
Apoptosis induction was also studied with the most active
compounds Prop-ASS-Ru3 and Prop-ASS by formation of
ssDNA in MDA-MB 231 and MCF-7 cells. As shown in
Figure 2, both compounds induced apoptosis time- and
dose-dependently, which was more pronounced, and in good
agreement with the proliferation experiments, after treat-
ment with Prop-ASS-Ru3. Similar effects were induced in
MCF-7 cells (data not shown).
Monolayers of the cells incubated for 24 h with 10 μM of the
complexes were stained with the dye and thereafter immedi-
ately analyzed under a fluorescence microscope. In accor-
dance with the results shown above, both compounds induced
growth inhibition and caused morphological changes (cell
blebbing and formation of apoptotic bodies), whereas Prop-
ASS-Ru3 was again more effective than Prop-ASS Co4 at
MDA-MB 231 (Figure 3) and MCF-7 (Figure S1, Support-
ing Information) cells.
Summary and Outlook
[( μ4-η2)-(Prop-2-ynyl)-2-acetoxybenzoate]dicobalthexacarbonyl
(Co-ASS) was synthesized in 1997 by Jung et al. among a
series of organometallic compounds and documented out-
standing in vitro activity. The molecule contains on the one
hand a organometal cluster and on the other hand in the
organic component aspirin, an effective inhibitor of cyclooxy-
genases. Both parts can interfere with intracellular pathways
leading to growth inhibition of tumor and leukemia cells. In
this study, we demonstrated that Co-ASS represents the ideal
composition of ASS and the [alkyne]Co2(CO)6 moiety. Higher
carbonyl cluster of cobalt or ruthenium did not increase the
activity. The use of iron cluster even completely terminated
the growth-inhibitory capacity.
An acridine orange-staining method was used for visuali-
zation of apoptotic processes in MDA-MB 231 and MCF-7 cells.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6895
and E/Z-penta-2,4-dien-1-ol14,15 were prepared by literature
procedures.
Fe2(CO)9 was prepared by a slightly modified literature
procedure28 using a quartz Schlenk tube, starting with 25 mL
of Fe(CO)5, 150 mL of glacial acetic acid, and 10 mL of acetic
anhydride (the latter was added to prevent the mixture contain-
ing too much water). A mercury-vapor lamp Philips HPL-N
250W was used for irradiation under cooling with a continuous
stream of air. Filtration, washing with water, ethanol, diethyl
ether, and subsequently drying in vacuo gave Fe2(CO)9 usually
in more than 90% yield. All complexations were carried out in
dry and degassed solvents under pure argon atmosphere.
General Method for the Preparation of 2-Acetoxybenzoic Acid
Esters. 2-Acetoxybenzoyl chloride (ASSCl, 10-20 mmol) dis-
solved in diethyl ether was added dropwise at 0 °C to an etherous
solution containing the respective alcohol (10-25 mmol) and
10 mL of pyridine as base. To ensure the completion of the
reaction, the solution was stirred for 2-3 h at room tempera-
ture. In a separating funnel, the organic layer was washed with
1 N HCl and afterward with saturated sodium bicarbonate
solution. The organic layer was dried over Na2SO4, and the
solvent was removed in vacuo. Purification was done by flash
column chromatography on silica gel.
(Prop-2-ynyl)-2-acetoxybenzoate (Prop-ASS). Prop-2-yn-1-ol,
1.30 g (23.2 mmol); ASSCl, 3.98 g (20.0 mmol). Column
chromatography: petroleum ether/diethyl ether = 3:1. Yield:
3.66 g (84%) as white crystals (mp 75.4 °C). 1H NMR (CDCl3,
400 MHz): δ = 2.35 (s, 3H, OdC-CH3), 2.53 (t, 4J = 2.4 Hz, 1H,
tC-H), 4.88 (d, 4J = 2.4 Hz, 2H, CH2-CtC), 7.11 (d, 3J =8.1Hz,
Figure 3. MDA-MB 231 cells stained by acridine orange and
observed under fluorescence microscopy (left, phase contrast
microscopy; right, fluorescence microscopy; 20ꢀ magnification).
(A) Cells without treatment (control: DMSO). (B) Cells incubated
with 10 μM of Prop-ASS-Ru3. (C) Cells incubated with 10 μM of
Prop-ASS-Co4.
3
4
1H, 30-H), 7.33 (ddd, J = 7.6 Hz, 3J = 7.6 Hz, J = 0.8 Hz,
1H, 50-H), 7.56 (ddd, 3J = 7.8 Hz, 3J = 7.6 Hz, 4J = 1.1 Hz, 1H,
40-H), 8.01 (dd, 3J = 7.8 Hz, 4J = 1.4 Hz, 1H, 60-H).
Furthermore, the assumed mode of action, the interference
in the arachidonic acid cascade, might be of minor relevance.
Prop-ASS-Ru3 and Prop-ASS-Co4 showed lower COX-
inhibitory effects but inhibited tumor-cell growth comparable
to Co-ASS. Vice versa, pentadiene-ASS-Ru as most potent
COX-inhibitor wasonlymarginally lessactive. Inthis context,
it is of great interest to look for the mode of action at the COX
enzymes and the acetylation profile compared to Co-ASS and
aspirin. These investigations are in progress and part of a
forthcoming paper.
(But-2-ynyl)-2-acetoxybenzoate (But-ASS). But-2-yn-1-ol, 1.55 g
(22.1 mmol); ASSCl, 4.01 g (20.2 mmol). Column chromatog-
raphy: petroleum ether/diethyl ether = 5:1. Yield: 4.18 g (89%)
1
as white crystals (mp 45.3 °C). H NMR (CDCl3, 400 MHz):
δ = 1.88 (t, 5J = 2.4 Hz, 3H, tC-CH3), 2.36 (s, 3H, OdC-
5
3
CH3), 4.84 (q, J = 2.4 Hz, 2H, O-CH2-Ct), 7.11 (dd, J =
8.1 Hz, 4J = 0.9 Hz, 1H, 30-H), 7.33 (ddd, 3J = 7.8 Hz, 3J=7.6 Hz,
4J = 0.9 Hz, 1H, 50-H), 7.54 (ddd, J = 7.9 Hz, J = 7.6 Hz,
3
3
4J=1.5 Hz, 1H, 40-H), 8.03 (dd, J=7.9 Hz, J=1.7 Hz, 1H,
3
4
60-H).
(But-2-yne-1,4-diyl)-bis(2-acetoxybenzoate) (Di-ASS). But-2-
yne-1,4-diol, 0.88 g (10.2 mmol); ASSCl, 4.21 g (21.2 mmol).
Column chromatography: n-hexane/ethyl acetate=5:1. Yield:
3.86 g (92%) as white crystals (mp 108.2 °C). 1H NMR (CDCl3,
400 MHz): δ=2.37 (s, 6H, OdC-CH3), 4.94 (s, 4H, O-CH2-
Ct), 7.12 (dd, 3J=8.1 Hz, 2H, 30-H), 7.30 (ddd, 3J=7.7 Hz, 3J=
7.5 Hz, 2H, 50-H), 7.59 (ddd, 3J=7.9 Hz, 3J=7.6 Hz, 4J=1.5 Hz,
2H, 40-H), 8.07 (dd, 3J=7.9 Hz, 4J=1.5 Hz, 2H, 60-H).
((E,Z)-Penta-2,4-dienyl)-2-acetoxybenzoate (pentadiene-ASS).
Penta-2,4-dien-1-ol, 1.69 g (20.1 mmol); ASSCl, 4.12 g (20.7 mmol).
Column chromatography: petroleum ether/diethyl ether (5:1).
Yield: 2.4 g (48%) cis and 1.8 g (37%) trans as mixture (colorless
oil). 1H NMR (CDCl3, 400 MHz): δ = 1.65-2.50 (m, 2H,
Experimental Section
Syntheses. General. Commercially available chemicals were
used without further purification. Solvents are purified by
distillation from an appropriate drying agent: tetrahydrofuran,
diethyl ether, toluene, n-pentane, and n-hexane were dried over
sodium/potassium alloy and distilled under argon atmosphere.
Pyridine was dried over KOH and dichloromethane over P2O5,
˚
and both were stored over molecular sieves (4 and 3 A,
respectively). Products were purified by flash chromatography
€
on silica gel (230-400 mesh, Merck). Melting points: 510 Buchi
(Flawil, Swiss) capillary melting point apparatus. IR spectra
3
€
(KBr pellets): Perkin-Elmer model 580 A (Rodgau-Jugesheim,
-CdCH2), 2.32 (s, 3H, OdC-CH3), 4.28-4.80 (d, 2H, J=6.0
1
Germany). H and 13C NMR: Avance DPX-400 spectrometer
Hz, -O-CH2-CHd), 5.00-6.36 (m, 3H, -HCdCH-CHdC), 7.09
(dd, 3J=8.1 Hz, 4J=1.0 Hz, 1H, 30-H), 7.30 (ddd, 3J=7.7 Hz, 3J=
7.5 Hz, 4J=1.0 Hz, 1H, 50-H), 7.54 (ddd, 3J=7.8 Hz, 3J=7.7 Hz,
4J=1.5 Hz, 1H, 40-H), 8.05 (dd, 3J=7.8 Hz, 4J=1.6 Hz, 1H, 60-H).
((2E,4E )-Hexa-2,4-dienyl)-2-acetoxybenzoate (hexadiene-ASS).
(2E,4E )-Hexa-2,4-dien-1-ol, 1.82 g (18.6 mmol);ASSCl, 3.98 g
(20.0 mmol). Column chromatography: petroleum ether/diethyl
ether=5:1. Yield: 3.77 g (78%) as colorless oil. 1H NMR (CDCl3,
400 MHz): δ=1.78 (d, 3J=6.8 Hz, 3H, dCH-CH3), 2.32 (s, 3H,
CO-O-CH3), 4.80 (d, 3J=6.7 Hz, 2H, -O-CH2-CHd), 5.74 (m,
2H, -CdCH-CH-Cd), 6.08(m, 1H, dCH-CH3), 6.28(m, 1H, CH2-
CHdC-), 7.11 (dd, 3J=8.1 Hz, 1H, 30-H), 7.30 (ddd, 3J=7.5 Hz,
3J=7.7, 4J=0.9 Hz, 1H, 50-H), 7.54 (ddd, 3J=7.9 Hz, 3J=7.6 Hz,
4J=1.7 Hz, 1H, 40-H), 8.02 (dd, 3J=7.7 Hz, 4J=1.7 Hz, 1H, 60-H).
(Bruker; Karlsruhe, Germany) at 400 MHz (1H) or 100 MHz
(13C), Avance III-700 spectrometer (Bruker) at 700 MHz (1H) or
175 MHz (13C) with TMS as internal standard. Elemental
€
analyses: Microlaboratory of the Freie Universitat Berlin on
Perkin-Elmer 240C. Purity of all synthesized substances was
confirmed by elemental analysis or HR-MS. All complexes
reported in the manuscript have a purity >95%. MS and
HR-MS spectra: Finnigan MAT 711 (EI, 70 eV), MAT CH7A
(EI, 80 eV, 3 kV), CH5DF (FAB, 80 eV, 3 kV), and Agilent ESI-
TOF 6210 (4 μL/min, 1 bar, 4000 V). Microplate reader:
FLASHscan S12 (AnalytikJena AG, Jena, Germany).
Synthesis. Co4(CO)12,16 Fe3(CO)12,35 trimethylamine-N-
oxide,36 2-acetoxybenzoyl chloride (ASSCl),37 ferrocenylcarbinol,31

6896 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19
Rubner et al.
General Method for the Preparation of ( μ4-η2)-(Alkynyl)-
tetracobaltdecacarbonyl. In a typical preparation, 410 mg of
dicobaltoctacarbonyl (Co2(CO)8, 1.2 mmol) were dissolved in
50 mL of dry n-hexane in a 100 mL Schlenk tube. The solution
was degassed to remove any oxygen and afterward fitted with a
bubble counter. The solution was refluxed for 5 h to produce
Co4(CO)12. After cooling to room temperature, 0.6 mmol of
the corresponding alkyne were added. Then the solution was
allowed to stir for three days with TLC control. The solvent was
removed under vaccuo and purified by flash chromatography
on silica gel.
1727 (CdO); 1608 (CdC); 1251, 1198 (C-O). 1H NMR (CDCl3,
400 MHz): δ=2.37 (s, 6H, OdC-CH3), 5.49 (s, 4H, -O-CH2-Ct),
7.12 (d, 3J=8.0 Hz, 2H, 30-H), 7.28 (dd, 3J=8.0 Hz, 3J=7.5 Hz,
3
4
2H, 50-H), 7.57 (ddd, J=7.9 Hz, 3J=7.6 Hz, J=1.2 Hz, 2H,
4
40-H), 8.07 (dd, 3J = 7.9 Hz, J = 1.5 Hz, 2H, 60-H). Anal.
(C31H18O17Ru) C, H.
[( μ3-η4)-(Penta-2,4-dienyl)-2-acetoxybenzoate]trirutheniumnona-
carbonyl (Pentadiene-ASS-Ru3). In a 500 mL Schlenk tube,
400 mg (0.63 mmol) of Ru3(CO)12 and 250 mg (1.0 mmol) of
pentadiene-ASS were dissolved in 350 mL of dry n-hexane. The
solution was degassed as described above and subsequently
refluxed for 18 h. TLC was used to control the progress of the
reaction. After cooling to room temperature, n-hexane was re-
moved in vacuo and the residue was purified by column chroma-
trography on silica gel using petroleum ether/diethyl ether=3:1.
Yield: 40 mg (8%) of light-yellow powder. IR (KBr, cm-1): 3061,
3019 (CdC-H); 2034, 1948 (Ru-CO); 1744 (CdO); 1610, 1592
(CdC); 1296, 1258 (C-O). Anal. (C23H14O13Ru3) C, H.
General Method for the Preparation of [η4-Alkadienyl]-
irontricarbonyl. In a typical preparation 1.1 g of Fe2(CO)9 (3 mmol)
and the corresponding diene (2 mmol) were dissolved in 50 mL of
dry diethyl ether in a 100 mL Schlenk tube. The solution was
degassed as described above and fitted with a bubble counter. The
solution was refluxed for 3 days with TLC control. After cooling to
room temperature, the solvent was removed in vaccuo and puri-
fied by flash chromatography on silica gel to receive light-yellow
powders.
[( μ4-η2)-(Prop-2-ynyl)-2-acetoxybenzoate]tetracobaltdecacarbonyl
(Prop-ASS-Co4). Prop-ASS: 130 mg (0.6 mmol). Column chro-
matography: petroleum ether/diethyl ether=10:1. Yield: 280 mg
(64%) of dark-blue oil. IR (KBr, cm-1): 2097, 2055, 2033
(terminal Co-CO), 1870 (bridged Co-CO), 1727, 1768 (CdO),
1629 (CdC), 1250, 1200 (C-O). 1H NMR (CDCl3, 700 MHz):
δ=2.38 (br, 3H, OdC-CH3), 5.03 (br, 2H, -O-CH2;), 6.10 (br,
1H, CtCH), 7.32 (br, 1H, 30-H), 7.63 (br, 1H, 50-H), 8.05 (br,
1H, 40-H), 8.42 (br, 1H, 60-H).
[( μ4-η2)-(But-3-ynyl)-2-acetoxybenzoate]tetracobaltdecacarbonyl
(But-ASS-Co4). But-ASS: 140 mg (0.6 mmol). Column chroma-
tography: petroleum ether/diethyl ether=15:1. Yield: 310 mg
(69%) of blue crystals. IR (KBr, cm-1): 2093, 2053, 2027
(terminal Co-CO), 1871 (bridged Co-CO), 1727 (CdO), 1629
1
(CdC), 1255, 1199 (C-O). H NMR (CDCl3, 400 MHz): δ=
2.28 (br, 3H, OdC-CH3), 2.76 (s, 3H, tC-CH3), 5.02 (br, 2H,
-O-CH2-CtC), 7.12 (br, 1H, 30-H), 7.30 (br, 1H, 50-H), 7.57
(br, 1H, 40-H), 8.00 (br, 1H, 60-H).
[η4-(Penta-2,4-dienyl)-2-acetoxybenzoate]irontricarbonyl
(Pentadiene-ASS-Fe). Pentadiene-ASS, 500 mg (2.0 mmol); Fe2-
(CO)9, 1.0 g (2.8 mmol). Column chromatrography: petroleum
ether/diethyl ether=10:1. Yield: 60 mg (10%) of light-yellow
powder. IR (KBr, cm-1): 3069, 3011 (CdC-H); 2051, 1978
(Fe-CO); 1769, 1722 (CdO); 1609, 1630 (CdC); 1292, 1198
[( μ4-η2)-(But-2-yne-1,4-diyl)-bis(2-acetoxybenzoate)]tetracobalt-
decacarbonyl (Di-ASS-Co4). Di-ASS: 240 mg (0.6 mmol). Col-
umn chromatography: petroleum ether/diethyl ether=5:1. Yield:
260 mg (48%), blue crystals. IR (KBr, cm-1): 2097, 2058, 2035
(terminal Co-CO), 1868 (bridged Co-CO), 1769, 1725 (CdO),
1607 (CdC), 1249, 1197 (C-O). 1H NMR (CDCl3): δ=2.35 (br,
6H, OdC-CH3), 5.49 (br, 4H, -O-CH2-Ct), 7.16 (br, 2H, 30-H),
7.35 (br, 2H, 50-H), 7.60 (br, 2H, 40-H), 8.05 (br, 2H, 60-H).
General Method for the Preparation of [( μ3-η2)-(Alkynyl)]-
trirutheniumnonacarbonyl. An excess of 0.2 mol of the corre-
sponding alkyne was treated with Ru3(CO)12 in 100 mL of dry
n-hexane in a 250 mL Schlenk tube. The solution was degassed
to remove any oxygen and afterward fitted with a bubble
counter. The solution was refluxed for 3 h. After cooling to
room temperature, n-hexane was removed under vaccuo and the
residue was purified by flash chromatography on silica gel.
[( μ3-η2)-(Prop-2-ynyl)-2-acetoxybenzoate]trirutheniumnona-
carbonyl (Prop-ASS-Ru3). Prop-ASS, 130 mg (0.6 mmol); Ru3-
(CO)12, 260 mg (0.41 mmol). Column chromatrography: petro-
leum ether/diethyl ether=5:1. Yield: 45 mg (14%) of orange oil.
IR (KBr, cm-1): 3071 (CtC-H); 2080, 2049, 2011 (Ru-CO);
1729 (CdO); 1609 (CdC); 1286, 1198 (C-O). 1H NMR (CDCl3,
400 MHz): δ=2.36 (s, 3H, OdC-CH3), 5.47 (br, 2H, -O-CH2-
Ct), 6.11 (br, 1H, CtC-H), 7.13 (d, 3J=8.1 Hz, 1H, 30-H), 7.31
(br, 1H, 50-H), 7.58 (br, 1H, 40-H), 8.10 (br, 1H, 60-H). Anal.
(C21H10O13Ru3) C, H.
1
2
3
(C-O). H NMR (CDCl3): δ = 0.45 (ddd, J = 9.4 Hz, J =
2.6 Hz, 4J=0.9 Hz, 1H, R-CdCH2), 1.06 (tdd, 3J=8.2 Hz, 3J=
5.8 Hz, 4J=0.9 Hz, 1H, -CH2-CH=), 1.81 (ddd, 2J=7.0 Hz,
3J=2.6 Hz, 4J=1.0 Hz, 1H, -CHdCH2), 2.30 (s, 3H, OdC-CH3),
4.27 (m, 2H, -O-CH2-Cd), 5.26 (m, 1H, HCdCH-CHdCH2),
5.42 (dd, 3J=8.2 Hz, 3J=4.8 Hz, 1H, HCdCH-CHdCH2), 7.06
(dd, 3J=8.1 Hz, 4J=1.1 Hz, 1H, 30-H), 7.28 (ddd, 3J=7.7 Hz,
3J=7.6 Hz, 4J=1.1 Hz, 1H, 50-H), 7.53 (ddd, 3J=7.8 Hz, 3J=
7.7 Hz, 4J=1.7 Hz, 1H, 40-H), 8.05 (dd, 3J=7.9 Hz, 4J=1.7 Hz,
1H, 60-H). Anal. (C17H14O7Fe) C, H.
[η4-(Hexa-2,4-dienyl)-2-acetoxybenzoate]irontricarbonyl
(Hexadiene-ASS-Fe). Hexadiene-ASS, 520 mg (2.0 mmol); Fe2-
(CO)9, 1.0 g (2.8 mmol). Column chromatrography: petroleum
ether/diethyl ether=10:1. Yield: 100 mg (13%) of light-yellow
powder. IR (KBr, cm-1): 3061, 3019 (CdC-H); 2046, 1969
(Fe-CO); 1768, 1706 (CdO); 1605 (CdC); 1295, 1196 (C-O).
1H NMR (CDCl3): δ=1.08 (ddq, 3J=5.8 Hz, 3J=2.6 Hz, 4J=
0.7 Hz, 1H, CdCH-CH3), 1.30 (m, 1H, CH2-CHd), 1.43 (d,
3J=3.6 Hz, 3H, CdCH-CH3), 2.33 (s, 3H, CO-O-CH3), 4.26 (m,
2H, O-CH2-CdC), 5.08 (dd, 3J = 8.4 Hz, 3J = 4.9 Hz, 1H,
HCdCH-CHdCH-CH3), 5.27 (dd, 3J=8.4 Hz, 3J=5.3 Hz, 1H,
3
4
HCdCH-CHdCH2), 7.11 (dd, J=8.1 Hz, J=1.1 Hz, 1H,
30-H), 7.31 (ddd, 3J=7.7 Hz, 3J=7.6 Hz, 4J=1.1 Hz, 1H, 50-H),
7.56 (ddd, 3J=7.8 Hz, 3J=7.7 Hz, 4J=1.7 Hz, 1H, 40-H), 8.05
(dd, 3J=7.9 Hz, 4J=1.7 Hz, 1H, 60-H). Anal. (C18H16O7Fe) C, H.
[( μ3-η2)-(But-3-ynyl)-2-acetoxybenzoate]triironnonacarbonyl
(But-ASS-Fe3) and Di-[( μ4-η4)-(But-3-ynyl)-2-acetoxybenzoate]-
triironoctacarbonyl (Di-(But-ASS)-Fe3). A 500 mL Schlenk flask
was charged with 1.5 g of Fe3(CO)12 (3 mmol) and 460 mg of But-
ASS (2 mmol). About 150 mL of dry n-pentane and 80 mL of dry
CH2Cl2 were added. This solution was cooled to -80 °C, and a
solution of 230 mg (3 mmol) of dehydrated trimethylamine-N-
oxide as a catalyst in 100 mL of CH2Cl2 was added. The solution
was degassed as described above and fitted with a bubble counter.
The solution was refluxed for 4 days. Solvents were removed under
vaccuo, and the residue was purified by column chromatography
with petroleum ether/diethyl ether (15:1).
[( μ3-η2)-(But-3-ynyl)-2-acetoxybenzoate]trirutheniumnonacarbonyl
(But-ASS-Ru3). But-ASS, 150 mg (0.65 mmol); Ru3(CO)12,
280 mg (0.44 mmol). Column chromatrography: petroleum ether/
diethyl ether=10:1. Yield: 60 mg (17%) of orange powder. IR
(KBr, cm-1): 2080, 2050, 2006 (Ru-CO); 1769, 1725 (CdO);
1608 (CdC); 1253, 1197 (C-O). 1H NMR (CDCl3, 400 MHz):
δ=2.36 (s, 3H, OdC-CH3), 2.66 (s, 3H, CtC-CH3), 5.49 (s, 2H,
3
3
-O-CH2-Ct), 7.14 (d, J=8.0 Hz, 1H, 30-H), 7.31 (dd, J=
7.6 Hz, 3J=7.6 Hz, 1H, 50-H), 7.58 (dd, 3J=7.5 Hz, 3J=7.1 Hz,
1H, 40-H),8.08(d,3J=7.6 Hz, 1H, 60-H).Anal.(C22H12O13Ru3) C,H.
[( μ3-η2)-(But-2-yne-1,4-diyl)-bis(2-acetoxybenzoate)]tri-
rutheniumnonacarbonyl (Di-ASS-Ru). Di-ASS, 410 mg (1.0 mmol);
Ru3(CO)12, 420 mg (0.66 mmol). Column chromatrography:
petroleum ether/diethyl ether=3:1. Yield: 35 mg (5.5%) of orange
crystals. IR (KBr, cm-1): 2086, 2058, 2023, 1947 (Ru-CO); 1767,

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 19 6897
But-ASS-Fe3. Yield: 20 mg (1.5%) as brown crystals. IR
measured in the plates for ssDNA apoptosis by the OD of the
wells analyzed with a modified MTT assay (EZ4U kit; Biomed-
ica, Vienna, Austria) as control for cellular biomass which was
set up in parallel. Apoptosis in the absence of compounds was
set at 1.
(KBr, cm-1): 2959, 2927, 2858 (C-H); 2061, 2026, 1986 (Fe-
CO); 1730 (CdO); 1636 (CdC); 1261, 1203 (C-O).
Di-(But-ASS)-Fe3. Yield: 15 mg (1.7%) of Di-(But-ASS)-Fe
as orange crystals. IR (KBr, cm-1): 2091, 2058, 2017, 1989 (Fe-
CO); 1734 (CdO); 1633 (CdC); 1261, 1200 (C-O).
For proliferation analysis, each well was exposed for the last
22 h of culture to 2 μCi of [3H-methyl]-thymidine (specific
activity 74 GBq/mmol, Hartmann Analytic, Braunschweig,
Germany). Cells were harvested using a semiautomated device
and [3H-methyl]-thymidine uptake expressed in counts per
minute was measured in a liquid scintillation counter
(Microbeta Trilux; Perkin-Elmer Life Sciences, Boston, MA).
Proliferation in the absence of the compounds was set at 100%.
Caspase-3 Activity Assay. Cells were cultured in black 96-well
microplates for 24 h (plating density 50000 cells/well) in a 5%
CO2/95% air atmosphere. Medium was then removed and
replaced by substance containing DMEM; control cells were
maintained in the absence of any compound. After 24 h of
treatment, cells were lysed (Cell Culture Lysis Reagent, Promega)
and incubated with 30 μM Ac-DEVD-AMC (biomol), a specific
caspase-3 substrate, which upon cleavage by active caspase-3
generates a highly fluorescent product, for 2 h at 37 °C. Fluores-
cence was assayed at excitation/emission wavelengths of 380/460 nm.
Caspase-3 activity in the absence of the compounds was set at 1.
Acridine Orange Staining. Each 620 μL of 7500 cells/mL
(MCF-7 and MDA-MB 231) were plated in 24-well flat bottom
plates at 37 °C in a 5% CO2/95% air atmosphere. After a three-
day culture, 10 μM of the test compounds were added in fresh
620 μL of medium and incubated for further 24 h. Thereafter,
cells were fixed with 200 μL of 1% glutardialdehyde/PBS and
stained with 200 μL of 2 μg/mL acridine orange/PBS (biomol)
for 2 min. Photos were taken with Zeiss Axiovert 40 CFL (λex 470/
40 nM, λem 525/50 nm) with 20ꢀ magnification.
Preparation of 1-((2-Acetoxybenzoyloxy)methyl)ferrocene
(Ferrocene-ASS). Synthesis was performed as described for
Prop-ASS. Ferrocenylcarbinol: 1.1 g (5.1 mmol), ASSCl: 1.07 g
(5.4 mmol). Column chromatrography: petroleum ether/diethyl
ether=5:1. Yield: 1.67 g (87%) as dark-yellow crystals (mp 118.2
°C). 1H NMR (CDCl3, 400 MHz): δ=2.17 (s, 3H, OdC-CH3),
4.18 (s, 5H, Fe-C5H5), 4.21 (t, 3J=1.8 Hz, 2H, Fe(C5H4), 3040-
3
H), 4.33 (t, J = 1.8 Hz, 2H, Fe(C5H4), 2050-H), 5.10 (s, 2H,
Fe(C5H4)-CH2-O), 7.07 (dd, 3J=8.1 Hz, 4J=1.1 Hz, 1H, 30-H),
7.29 (ddd, 3J=7.7 Hz, 3J=7.6 Hz, 4J=1.1 Hz, 1H, 50-H), 7.54
(ddd, 3J=7.8 Hz, 3J=7.6 Hz, 4J=1.6 Hz, 1H, 40-H), 8.02 (dd,
3J=7.9 Hz, 4J=1.7 Hz, 1H, 60-H). Anal. (C20H18O4Fe) C, H.
Biological Methods. Cytotoxicity Experiments. The human
MCF-7 and MDA-MB 231 breast cancer cell lines as well as the
HT-29 colon cancer cell line were obtained from the American
Type Culture Collection (ATCC, USA). Cell line banking and
quality control were performed according to the seed stock
concept reviewed by Hay.38 All three cell lines were maintained
in L-glutamine and 4.5 g/L D-glucose containing Dulbecco’s
Modified Eagle Medium (DMEM; PAA, Pasching, Austria)
without phenol red, supplemented with 5% fetal calf serum
(FCS; Biochrom, Berlin, Germany) using 25 cm2 culture flasks
€
(Sarstedt, Nurnberg, Germany) in a humidified atmosphere at
37 °C. The MCF-7 cell line was passaged weekly after treatment
with trypsin (0.05%) and ethylenediaminetetraacetic acid
(0.02%; EDTA; Boehringer, Mannheim, Germany), whereas
HT-29 and MDA-MB-231 where passaged twice per week.
Each 100 μL of 7500 cells/mL (MCF-7), 2850 cells/mL (HT-
29), or 7500 cells/mL (MDA-MB 231) were incubated in 96-well
plates at 37 °C in a 5% CO2/95% air atmosphere for 72 h with
the test compound of various concentration. One plate was used
for the determination of the initial cell biomass. The medium
was removed and the cells were fixed by a 20-30 min incubation
with 100 μL of glutardialdehyde solution (0.5 mL of glutardial-
dehyde þ12.5 mL of phosphate-buffered saline (PBS), pH 7.4).
The solution in the wells was sucked off, 180 μL of PBS, pH 7.4,
were added, and the plate was stored at 4 °C until further
treatment. In the “experimental” plates, the medium was re-
placed with medium containing the drugs in graded concentra-
tions (eight replicates). After further incubation for 96 h, these
plates were treated as described above. The cell biomass was
determined by crystal violet staining according to the following
procedure: the PBS, pH 7.4 was removed, 100 μL of a 0.02 M
crystal-violet solution were added, and plates were incubated for
30 min at room temperature, washed three times with water, and
incubated on a softly rocking rotary shaker with 180 μL of
ethanol (70%) for further 3-4 h. Absorption was recorded at
590 nm using a microplate reader (FLASHscan S12). The mean
absorption of the initial cell biomass plate was subtracted from
the mean absorption of each experiment and control. The
corrected control was set at 100%. IC50 value was determined
as that concentration causing 50% inhibition of cell growth and
calculated as mean of at least two independent experiments.
Analysis of Proliferation and Apoptosis. In each experiment,
100 μL of 7500 cells/mL (MCF-7 and MDA-MB 231) were
plated in 96-well flat bottom plates at 37 °C in a 5% CO2/95%
air atmosphere. After a three-day culture various concentra-
tions (5 and 10 μM) of test compounds were added in
100 μL and further incubated for 24, 48, and 72 h. Thereafter, cul-
tures were analyzed for cellular proliferation using [3H-methyl]-
thymidine uptake and apoptosis induction using the ssDNA
apoptosis kit (Chemicon International, Hofheim, Germany)
according to the manufacturer’s instructions. Apoptosis rate
was evaluated by dividing the optical density (OD) of the wells
Inhibition of COX-Enzymes. The inhibition of isolated ovine
COX-1 and human recombinant COX-2 at 10 μM of the respective
compounds was determined by ELISA (COX Inhibitor Screening
Assay; Cayman Chemical Company, Ann Arbor, MI). The experi-
ments were performed according to the manufacturer’s instruc-
tions. Absorption was measured at 415 nm (FLASHscan S12).
PGE2-Assay. MDA-MB 231 cells were grown in 24-well
microplates in a 5% CO2/95% air atmosphere until at least
70% confluency. The medium was then removed and replaced
by fresh DMEM containing the respective substances (10 μM);
control cells were maintained in the absence of any compound.
The cells were incubated for 23 h followed by the addition of
100 μM of arachidonic acid. Twenty-four h after the addition of
the corresponding substances, the drug-containing medium
with the including prostaglandin E2 was removed and tested
by ELISA (Prostaglandin E2 EIA Kit-Monoclonal; Cayman
Chemicals). Experiments were performed according to the manu-
facturer’s instructions. Absorption was measured at 415 nm
(FLASHscan S12).
Acknowledgment. The financial support of DFG;Deutsche
Forschungsgemeinschaft (project: FOR 630) is greatly appre-
€
ciated. We especially thank Evelina Fogelstrom for reviewing
this paper.
Supporting Information Available: Additional spectroscopic
data of title compounds; elemental analyses of all complexes;
acridine orange staining of MCF-7 cells. This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
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