Acetylenehexacarbonyldicobalt complexes, a novel class of antitumor drugs

Article abstract of DOI:10.1016/S0020-1693(00)00139-0

Acetylenehexacarbonyldicobalt complexes were synthesized and tested for antitumor activity. The MCF-7 and MDA-MB-231 mammary tumor cell lines and the LNCaP/FGC prostate carcinoma cell line were used as in vitro models. The structural evaluation was performed by IR and NMR spectroscopy and revealed a change of the linear acetylene core to a structure comparable to Z-olefins after coordination to the cobalt centers. In cell culture experiments the strongest effects were found for hexacarbonyl[2-propinylacetylsalicylate]dicobalt (10), which was more active than cisplatin on the human mammary tumor cell lines MCF-7 and MDA-MB-231 in each concentration tested (a 5 μM concentration of this compound even caused cytocidal effects). In contrast to this, 10 influenced the growth of the LNCaP/FGC cells only marginally, even in the highest concentration. The mode of action of the complexes tested is unknown. As the cobalt complexes show strong antiproliferative effects and their ligands do not it could be unambiguously demonstrated that complex formation is essential to achieve cytotoxic effects. (C) 2000 Elsevier Science S.A.

Full text of DOI:10.1016/S0020-1693(00)00139-0

www.elsevier.nl/locate/ica
Inorganica Chimica Acta 306 (2000) 6–16
Acetylenehexacarbonyldicobalt complexes, a novel class of
antitumor drugs
Kathrin Schmidt a, Manfred Jung b, Roland Keilitz a, Beate Schnurr a, Ronald Gust a,*
a Institut fu¨r Pharmazie, Freie Uni6ersita¨t Berlin, Ko¨nigin-Luise-Strasse 2+4, D-14195 Berlin, Germany
b Institut fu¨r Pharmazeutische Chemie, Westfa¨lische Wilhelms-Uni6ersita¨t Mu¨nster, Hittorfstrasse 58-62, D-48149 Mu¨nster, Germany
Received 28 December 1999; accepted 24 March 2000
Abstract
Acetylenehexacarbonyldicobalt complexes were synthesized and tested for antitumor activity. The MCF-7 and MDA-MB-231
mammary tumor cell lines and the LNCaP/FGC prostate carcinoma cell line were used as in vitro models. The structural
evaluation was performed by IR and NMR spectroscopy and revealed a change of the linear acetylene core to a structure
comparable to Z-olefins after coordination to the cobalt centers. In cell culture experiments the strongest effects were found for
hexacarbonyl[2-propinylacetylsalicylate]dicobalt (10), which was more active than cisplatin on the human mammary tumor cell
lines MCF-7 and MDA-MB-231 in each concentration tested (a 5 mM concentration of this compound even caused cytocidal
effects). In contrast to this, 10 influenced the growth of the LNCaP/FGC cells only marginally, even in the highest concentration.
The mode of action of the complexes tested is unknown. As the cobalt complexes show strong antiproliferative effects and their
ligands do not it could be unambiguously demonstrated that complex formation is essential to achieve cytotoxic effects. © 2000
Elsevier Science S.A. All rights reserved.
Keywords: Antitumor activity; Cobalt complexes; Carbonyl complexes
1. Introduction
As an approach for the treatment of hormone depen-
dent tumors (e.g. the estrogen receptor positive breast
cancer), metal complexes of estradiol derivatives were
synthesized [7]. Out of this class of compounds the
hexacarbonyl[ethinylestradiol]dicobalt complex was
also used to monitor the binding of steroids to the
estrogen receptor [8]. The formation of an a-cation in
the vicinity of the Co2(CO)6 moiety enabled the com-
plex to bind covalently to nucleophilic centers in the
hormone binding site of the estrogen receptor.
This strategy of drug design induced us to synthesize
hexacarbonyldicobalt complexes as cytostatics. The first
results were published previously [9] and demonstrated
the tumor inhibiting properties of acetylenehexacar-
bonyldicobalt complexes. In this study the antiprolifer-
ative effects depended strongly on the structure of the
bound acetylene ligand. On the H2981 lung adeno-
carcinoma cell line, treatment with 10 mM of hexacar-
bonyl[2-propinylacetylsalicylate]dicobalt (10) and hexa-
carbonyl[2-propinylsalicylate]dicobalt (11) led to an
80–90% inhibition of the [3H]-thymidine incorpora-
Among the multitude of drugs used in the treatment
of cancer cis-diamminedichloroplatinum(II) (cisplatin)
is established as a cytotoxic1 agent. Cisplatin has shown
to be very active against testicular and ovarian tumors,
tumors of the head and neck as well as against bladder
tumors [1,2]. However, it is not or is insufficiently active
against tumors of the gastrointestine, the lung and the
mammary. Since these tumors account for the major
part of cancer mortality today the chemical and clinical
search for new selectively acting cisplatin derivatives or
new tumor inhibiting metal complexes [3–6] goes on.
* Corresponding author. Tel.: +49-30-838 53272; fax: +49-30-
838 53854.
E-mail address: rgust@zedat.fu-berlin.de (R. Gust).
1 Cytotoxic effect: being damaging to the cells. Cytocidal effect:
reduction of initial cell mass, cells are being killed. Cytostatic effect:
prevention or considerable inhibition of cell proliferation.
0020-1693/00/$ - see front matter © 2000 Elsevier Science S.A. All rights reserved.
PII: S0020-1693(00)00139-0

K. Schmidt et al. / Inorganica Chimica Acta 306 (2000) 6–16
7
tion. To achieve the same effect hexacarbonyl[N-2-
propinylbenzothiazolidine - 3 - one - 1,1 - dioxide]dicobalt
(9) had to be used in a fivefold higher concentration.
Due to these positive results we tested these
acetylenehexacarbonyldicobalt complexes also on fur-
ther tumor cell lines (human breast cancer cell lines
MCF-7 and MDA-MB-231, human prostate cancer cell
line LNCaP/FGC) and elucidated their structure in
detail. In this paper we present these results along with
the synthesis of new compounds (see Chart 1).
mmol) of 4-dimethylaminopyridine were stirred in 15
ml of dry CH2Cl2 over night. The mixture was filtered,
the precipitate was rinsed with CH2Cl2 and the com-
bined filtrates were evaporated. The resulting oil was
chromatographed with ethyl 1:5 acetate–hexane. The
first fraction contained the product. Yield: 410 mg
(12%); colorless crystals; m.p. 43°C. IR (KBr): w¯ =1740
cm−1 (CO), 1700 cm−1 (CO). MS (70 eV): m/z (%)=
1
232 (6) [M+], 121 (100). H NMR (CDCl3): l=1.88 (t,
(Chart 1)
2. Experimental
5J=2.2 Hz, 3H, CꢀCH3), 2.38 (s, 3H, COCH3), 4.85 (q,
5J=2.2 Hz, 2H, OCH2), 7.09 (dd, J=8.0 Hz, J=1.2
2.1. General procedure
3
4
Hz, 1H, 3%-H), 7.33 (ddd, 3J=7.5 Hz, 3J=7.8 Hz,
Melting point: uncorrected. Elemental analysis:
Perkin–Elmer. IR (KBr): Matson Genesis. H NMR:
3
3
4J=1.2 Hz, 1H, 5%-H), 7.58 (ddd, J=8.0 Hz, J=7.5
Hz, 4J=1.7 Hz, 1H, 4%-H), 8.07 (dd, 3J=7.8 Hz,
4J=1.7 Hz, 1H, 6%-H). 13C NMR (CDCl3): l=3.73
(CꢁCꢀCH3), 21.03 (COCH3), 53.59 (OCH2), 83.04
(CꢁCꢀCH3), 83.69 (CꢁCꢀCH3), 122.95 (CꢀCOO),
123.94 (C-3), 126.12 (C-5), 132.12 (C-6), 134.18 (C-4),
150.78 (CꢀOAc), 163.99 (COOR), 169.80 (MeCO).
Anal. Calc. for C13H12O4: C, 67.2; H, 5.22. Found: C,
67.1; H, 5.26%.
1
Varian Gemini 200 (200 MHz). 13C NMR: Varian
Gemini 200 (50.29 MHz). MS: Varian MAT 44S,
Finnigan MAT 312. Flash chromatography: silica gel
60, 230–400 mesh (Merck). (S)-Naproxen and acetyl-
salicylic acid were purchased from Sigma. Dicobaltoc-
tacarbonyl (Fluka, moistened with 5–10% hexane) was
used as purchased and presumed to be of 95% purity.
THF was distilled from LiAlH4 under nitrogen prior to
,
use. CH2Cl2 was dried over molecular sieves (4 A). The
syntheses of 7, 9, 10 and 11 were published previously
[9,10].
2.2.2. (S)-(2-Propinyl)-2-(6-methoxy-2-naphthyl)-
propionate (5)
A total of 921 mg (4 mmol) of (S)-naproxen (4), 908
mg (4.4 mmol) of dicyclohexylcarbodiimide, 0.26 ml
(252 mg, 4.5 mmol) of propargyl alcohol and 61 mg
(0.5 mmol) of 4-dimethylaminopyridine were stirred in
15 ml of dry CH2Cl2 over night. The mixture was
filtered, the precipitate was rinsed with CH2Cl2 and the
combined filtrates were evaporated. The resulting oil
2.2. Syntheses of the alkyne ligands
2.2.1. (2-Butin-1-yl)acetylsalicylate (2)
A total of 900 mg (5 mmol) of acetylsalicylic acid (1),
1.07 g (5.2 mmol) of dicyclohexylcarbodiimide, 0.41 ml
(386 mg, 5.5 mmol) of 2-butine-1-ol and 61 mg (0.5

8
K. Schmidt et al. / Inorganica Chimica Acta 306 (2000) 6–16
was chromatographed with 1:3 ethyl acetate–hexane
and the solid obtained was recrystallized from hexane.
Yield: 870 mg (81%); colorless crystals; m.p. 69–70°C.
IR (KBr): w¯ =1730 cm−1 (CO). MS (70 eV): m/z
(%)=268 (47) [M+], 185 (100). 1H NMR (CDCl3):
2.3.2. Hexacarbonyl[(S)-(2-propinyl)-2-
(6-methoxy-2-naphthyl)propionate]dicobalt (6)
General method for acetylenehexacarbonyldicobalt
complexes; 268 mg (1 mmol) 5. After chromatography
with 1:10 ethyl acetate–hexane and evaporation a
brown oil was obtained. Yield: 310 mg (56%). MS (70
eV): m/z (%)=470 (62) [M+−3CO], 442 (6) [M+−
4CO], 414 (64) [M+−5CO], 386 (100) [M+−6CO]. IR
3
4
l=1.59 (d, J=7.1 Hz, 3H, CH3), 2.43 (t, J=2.5 Hz,
3
1H, CH), 3.89 (s, 3H, OCH3), 3.98 (q, J=7.1 Hz, 1H,
2
4
ArꢀCH), 4.60 (dd, 1H, J=15.6 Hz, J=2.5 Hz, 1H,
2
4
OCH2), 4.73 (dd, 1H, J=15.6 Hz, J=2.5 Hz, 1H,
1
(KBr): w¯ =2097, 2058, 2029 cm−1 (CoꢀCO). H NMR
3
OCH2), 7.11–7.17 (m, 2H, ArꢀH), 7.40 (dd, J=8.5
3
(CDCl3): l=1.62 (d, J=6.3 Hz, 3H, CH3), 3.92 (s,
4
Hz, J=1.9 Hz, 1H, ArꢀH), 7.67–7.72 (m, 3H, ArꢀH).
4H, ArꢀCH and OCH3), 5.12 (d, 1H, 2J=14 Hz,
13C NMR (CDCl3): l=18.61 (CHCH3), 45.25
(ArꢀCH), 52.33 (OCH2), 55.36 (OCH3), 74.94
(CꢁCꢀH), 77.68 (CꢁCꢀH), 105.69 (Cꢀ7), 119.08 (C-5%),
126.09 and 126.20 (C-1% and 3%), 127.29 (C-4%), 128.98
(C-8%a), 129.35 (C-8%), 133.82 (C-2%), 135.19 (C-4%a),
157.77 (COMe), 173.88 (CO). Anal. Calc. for
C17H16O3: C, 76.1; H, 6.02. Found: C, 75.6; H, 6.23%.
2
OCH2), 5.40 (d, 1H, J=14 Hz, OCH2), 6.00 (s, 1H,
CꢀH), 7.08–7.16 (m, 2H, ArꢀH), 7.40–7.45 (m, 1H,
ArꢀH), 7.69 (s, 3H, ArꢀH). 13C NMR (CDCl3): l=
18.51 (CH3), 45.46 (ArꢀCH), 55.39 (OCH3), 65.49
(OCH2), 72.04 (CꢁCꢀH), 88.66 (OCH2ꢀC), 105.73 (C-
7), 119.06 (C-5), 126.25 (C-1 and C-3), 127.31 (C-4),
129.08 (C-8a), 129.34 (C-8), 133.89 (C-2), 135.43 (C-
4a), 157.78 (COMe), 174.47 (CO), 199.0 (CoꢀCO).
Anal. Calc. for C23H16Co2O9: C, 49.8; H, 2.92. Found:
C, 50.1; H, 3.06%.
2.3. General method for the preparation of acetylene-
hexacarbonyldicobalt complexes
A total of 1 mmol of the alkyne was dissolved in 10
ml of dry THF in an oven dried flask which had been
purged with nitrogen and 377 mg (1.05 mmol) of
dicobaltoctacarbonyl (Fluka, 95%) were added. Evolu-
tion of carbon monoxide immediately started upon
addition. The reaction mixture was stirred for 6 h at
room temperature (r.t.). 1 g of silica gel 60 was then
added and the mixture was evaporated to dryness. The
dark powder was subjected to flash column chromatog-
raphy with the requisite solvent mixture. Dark colored
product containing fractions were evaporated to dry-
ness. Unless stated otherwise the compounds solidified
and were of analytical purity without recrystallization.
2.3.3. [(R,S)-1-(4-Fluorophenyl)-1-phenyl-4-
pyrrolidinyl-2-butine-1-ol]hexacarbonyldicobalt (8)
General method for acetylenehexacarbonyldicobalt
complexes; 309 mg (1 mmol) 7. After chromatography
with ethyl acetate/hexane and evaporation brown–red
crystals were obtained. Yield: 390 mg (66%); m.p. 125–
126°C. MS (70 eV): m/z (%)=511 (18) [M+−3CO],
483 (39) [M+−4CO], 455 (16) [M+−5CO], 427 (15)
[M+−6CO], 84 (100). IR (KBr): w¯ =2091, 2052, 2035,
2024, 2013 cm−1 (CoꢀCO). 1H NMR (CDCl3): l=1.92
(s, 4H, NCH2CH2), 2.86 (s, 4H, NCH2CH2), 3.91 (s,
2H, CH2N), 6.97–7.05 (m, 2H, ArꢀH), 7.21–7.35 (m,
3H, ArꢀH), 7.59–7.70 (m, 4H, Ar-H), 8.04 (s, 1H,
OH). Anal. Calc. for C26H20Co2FNO7: C, 52.5; H, 3.39;
N, 2.35. Found: C, 52.7; H, 3.35; N, 2.61%.
2.3.1. [(2-Butin-1-yl)acetylsalicylate]hexacarbonyl-
dicobalt (3)
General method for acetylenehexacarbonyldicobalt
complexes; 232 mg (1 mmol) 2. After chromatography
with 1:10 ethyl acetate–hexane and evaporation a
brown oil was obtained. Yield: 180 mg (35%). MS (70
eV): m/z (%)=462 (8) [M+−2CO], 434 (41) [M+−
3CO], 406 (26) [M+−4CO], 378 (19) [M+−5CO], 350
[M+−6CO], 306 (100). IR (KBr): w¯ =2093, 2054, 2025
cm−1 (CoꢀCO). 1H NMR (CDCl3): l=2.37 (s, 3H,
COCH3), 2.66 (s, 3H, ꢁCꢀCH3), 5.49 (s, 2H, OCH2),
2.4. Biological methods
2.4.1. Cell culture
The human MCF-7 and MDA-MB-231 breast cancer
cell lines as well as the LNCaP/FGC 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 [11]. The MCF-7 cells were main-
3
7.13 (d, J=7.7 Hz, ArꢀH), 7.30–7.34 (m, 1H, ArꢀH),
7.54–7.59 (m, 1H, ArꢀH), 8.09 (1H, ArꢀH). 13C NMR
(CDCl3): l=20.28 and 20.99 (ꢁCꢀCH3 and COCH3),
65.33 (OCH2), 89.93 (CꢁCꢀCH3), 93.37 (CꢁCꢀCH3),
122.31 (CꢀCOO), 123.98, 126.02, 131.52 and 134.26
(ArꢀCH), 151.31 (CꢀOAc), 163.66 and 169.74
tained in
Germany), supplemented with NaHCO3 (2.2 g l−1),
sodium pyruvate (110 mg l−1), gentamycin (50 mg l−1
L-glutamine containing Eagle’s MEM (Sigma,
)
and 10% fetal calf serum (FCS; Gibco, Germany) using
75 cm2 culture flasks in a humidified atmosphere (95%
air/5% CO2) at 37°C. The MDA-MB-231 cells (Mc-
Coy’s 5A medium supplemented with NaHCO3 (2.2 g
(COOR),
199.2
(CoꢀCO).
Anal.
Calc.
for
C19H12Co2O10: C, 44.0; H, 2.34. Found: C, 44.1; H,
2.52%.

K. Schmidt et al. / Inorganica Chimica Acta 306 (2000) 6–16
9
l
l
−1), sodium pyruvate (110 mg l−1), gentamycin (50 mg
−1) and 5% FCS) and the LNCaP/FGC cells (
-glu-
staining technique as described earlier [11,12]. The ef-
fectiveness of the complexes is expressed as corrected
percentage T/Ccorr values according to the following
equations: cytostatic effect: T/Ccorr [%]=[(T−Co)/
(C−Co)]×100, where T (test) and C (control) are the
optical densities at 578 nm of the crystal violet extract
of the cells in the wells (i.e. the chromatin-bound crystal
violet extracted with ethanol 70%), and Co is the optical
density of the crystal violet extract immediately before
treatment. Cytocidal effect: ~ [%]=[(T−Co)/Co]×100.
For the automatic estimation of the optical density of
the crystal violet extract in the wells a Microplate EL
309 Autoreader was used.
L
tamine containing RPMI 1640, supplemented with
NaHCO3 (2.0 g l−1), gentamycin (50 mg l−1) and 7.5%
FCS) were maintained under the same conditions. The
cell lines were passaged weekly after treatment with
trypsin (0.05%)/ethylenediaminetetraacetic acid (0.02%;
EDTA; Boehringer, Germany). Mycoplasma contami-
nation was routinely monitored and only mycoplasma-
free cultures were used.
2.4.2. In 6itro chemosensiti6ity assays
The in vitro testing of the cobalt complexes for
antitumor activity was carried out on exponentially
dividing human cancer cells according to a previously
published microtiter assay [12,13]. Briefly, in 96-well
microtiter plates, 100 ml of a cell suspension at 7700
cells ml−1 culture medium (MCF-7) at 3200 cells ml−1
(MDA-MB-231) and at 2700 cells ml−1 (LNCaP/
FGC), respectively, were plated into each well and
incubated at 37°C for 3 or 5 days, (LNCaP/FGC) in a
humidified atmosphere (5% CO2). By addition of an
adequate volume of a stock solution of the respective
compound (solvent: DMF) to the medium the desired
3. Results and discussion
3.1. Synthesis and structural characterization
3.1.1. Synthesis
For the syntheses of the drugs 3, 6 and 8 we used the
course of reaction described by Jung et al. [9] (see Chart
2).
In the first step acetylsalicylic acid (1) was reacted
with 2-butine-1-ol to yield (2-butin-1-yl)acetylsalicylate
Chart 2
test concentration was obtained. For each test concen-
tration and for the control, which contained the corre-
sponding amount of DMF, 16 wells were used. After
the proper incubation time the medium was removed,
the cells were fixed with a glutardialdehyde solution
and stored under phosphate buffered saline (PBS) at 4
°C. Cell biomass was determined by a crystal violet
(2) while esterification of (S)-naproxen (4) with propar-
gyl alcohol resulted in (S)-(2-propinyl)-2-(6-methoxy-2-
naphthyl)propionate (5) (see Chart 2).
For coordination, the acetylenes were stirred with
Co2(CO)8 at r.t. The separation of the acetylenehexacar-
bonyldicobalt complexes from their reaction mixtures
was performed by column chromatography on SiO2.

10
K. Schmidt et al. / Inorganica Chimica Acta 306 (2000) 6–16
Fig. 1. X-ray structure of hexacarbonyl[diphenylacetylene]dicobalt [14] (left) and a low-energy conformation of 10 (right) generated by Sybyl 6.6
(Tripos, Inc.) using the Tripos force field and Gasteiger–Huckel charges.
3.1.2. Structural characterization
split by long range couplings (see Table 1 and data in
the experimental part). After coordination to cobalt the
signals are shifted to lower field (CꢀHA, CꢀHC about
0.5–0.8 ppm and CꢀHB about 3.5–3.8 ppm) and are
simplified due to the loss of the long range couplings.
These results indicate a reduction of the triple bond
character of the acetylenes and the change of the linear
geometry to that of a Z-olefin [17].
For the characterization of the acetylenehexacar-
bonyldicobalt complexes and the evaluation of their
spatial structure IR and NMR spectroscopy were used.
Due to the binding to the cobalt atoms the spatial
structure of the acetylene as well as the binding mode
of the carbonyl ligands changed. The triple bond of the
acetylene moiety with two perpendicular oriented p-or-
bitals makes the coordination to Co2(CO)8 possible
accompanied by elimination of two CO ligands from
their coordination places. The binding mode should be
comparable to that of hexacarbonyl[dipheny-
lacetylene]dicobalt [14]. Fig. 1 represents the X-ray
structure of this complex together with the low energy
conformation of 10.
The coordination changes not only the geometry of
the ligand but also the electronic environment at C2 and
C3, well documented by the resonances in the 13C
In both complexes the XꢀCꢁCꢀX moiety of the
acetylene is oriented to the CoꢀCo-axis in an angle of
90° with p-bonds to the cobalt atoms. Consequently,
the triple bond character is lowered. Compared to the
free acetylene the CꢀC-distance increased from about
,
1.2 to about 1.5 A. Furthermore, the linearity of the
alkyne is lost (XꢀCꢁCꢀX dihedral angleB180°).
The binding mode of the carbonyl ligands in the
hexacarbonyldicobalt complexes was evaluated by IR-
spectroscopy. The comparison of the spectra of the
complexes 3, 6, 8–11 with that of Co2(CO)8 indicates
that the CO ligands in the complex are exclusively
terminally oriented. In Co2(CO)8 two carbonyl groups
are bridged between the metal atoms. In accordance
with this, the IR-spectra exhibits stretching vibrations
(w¯CO) at 1832 and 1846 cm−1 for the bridged CO.
From the asymmetrical structure of the acetylenehexa-
carbonyldicobalt complexes result six different stretch-
ing vibrations [15]. In the case of complex 10 (see Fig.
2), the CO absorptions are well separated at 2008, 2018,
2033, 2040, 2059 and 2097 cm−1
.
1
The coordination to cobalt changes the H and 13C
NMR resonances of the acetylenes characteristically
1
[16]. In the H NMR spectra of the free acetylenes the
resonances for CꢁCꢀHB (l=2.39–2.56), CꢁCꢀCH2A
(l=3.43–4.95) or CꢁCꢀCH3C (l=1.88) protons are
Fig. 2. IR spectra of Co2(CO)8 and 10.

K. Schmidt et al. / Inorganica Chimica Acta 306 (2000) 6–16
11
Table 1
NMR dataa of acetylenehexacarbonyldicobalt complexes
NMR-spectra [18]. The chemical shift of the 13C atoms
depends on the configuration, as well as on the adjacent
substituents.
In case of the acetylenes presented in this paper, the
different substituents at the sp-carbons cause separate
resonances for C2 and C3. In the 13C NMR spectra of
Fig. 3. Effect of cisplatin, CoCl2 and Co2(CO)8 (0.5, 1.0, 5.0 mM) on the proliferation of human MCF-7 cells.

12
K. Schmidt et al. / Inorganica Chimica Acta 306 (2000) 6–16
Fig. 4. Effect of 3, 6, 8 and 9 (0.5, 1.0, 5.0 mM) on the proliferation of human MCF-7 cells.
the free ligands the separation amounts to Dl1=
l(C2)−l(C3)=0.06–4.56 (see Table 1). This effect is
more marked for the hexacarbonyldicobalt complexes
6, 9–11 (Dl2=13.14–16.62, see Table 1). The p-bond
to the metal atom leads at C2 to a strong deshielding
and therefore to a low-field shift of this signal. The
magnitude of this ‘coordination effect’ is expressed by
DDl=Dl2−Dl1 and amounts to 10.38–15.53 (see
Table 1). This means, that the CꢁC core of the
acetylene is more polarized in the coordinated molecule
than in the free state. Complexes 3 and 8 show the same
effect, however, to a smaller extent (DDl (3)=2.79;
DDl (8)=6.24).
3.2. Antiproliferati6e effects
The complexes 3, 6, 8–11 were tested on the mam-
mary carcinoma cell lines MCF-7 and MDA-MB-231
and the LNCaP/FGC prostate cancer cell line. As
reference cisplatin was used. Figs. 3–5 present the
results of the tests on the MCF-7 cell line. Cisplatin
reduced the growth of the tumor cells dependent on the

K. Schmidt et al. / Inorganica Chimica Acta 306 (2000) 6–16
13
concentration used. In a concentration of 5 mM it sup-
pressed the cell proliferation completely (T/Ccorr=0%,
see Fig. 3). The antiproliferative potency of the
acetylenehexacarbonyldicobalt complexes depends on
the structure of the coordinated alkyne. [(2-Butin-1-
yl)acetylsalicylate]hexacarbonyldicobalt (3) and hexa-
carbonyl[(S)-(2-propinyl)-2-(6-methoxy-2-naphthyl)-
propionate]dicobalt (6) reduced the cell growth only
slightly in the highest concentration used (Fig. 4). An-
tiproliferative effects (T/Ccorr=50%) were determined
by [1-(4-fluorophenyl)-1-phenyl-4-pyrrolidinyl-2-butine-
1-ol]hexacarbonyldicobalt (8) in a concentration of 5
mM. The most active compounds were the complexes
9–11. After incubation for about 125 h hexacar-
bonyl[N-2-propinylbenzothiazolidine-3-one-1,1-dioxide]-
dicobalt (9) reached a T/Ccorr of 8% (Fig. 4) while the
complexes
hexacarbonyl[2-propinylacetylsalicylate]-
dicobalt (10) and hexacarbonyl[2-propinylsalicylate]-
dicobalt (11) caused cytocidal effects in a concentration
of 5 mM (see Fig. 5). Furthermore, complex 10 was
more active than cisplatin in all concentrations used
(compare Figs. 3 and 5).
The same trends in activity were found for the com-
plexes on the MDA-MB-231 cell line. The complexes 3
and 6 influenced the growth of the MDA-MB-231 cells
only marginally (T/Ccorr:75% at 5 mM) (not shown),
while 8 and 9 caused a 50% reduction of the prolifera-
tion in a concentration of 5 mM (Fig. 6). Analogously
Fig. 5. Effect of 11b, 11a, 11 and 1, 10a, 10 (0.5, 1.0, 5.0 mM) on the proliferation of human MCF-7 cells.

14
K. Schmidt et al. / Inorganica Chimica Acta 306 (2000) 6–16
Fig. 6. Effect of 8, 9, 10 and 11 (0.5, 1.0, 5.0 mM) on the proliferation of human MDA-MB-231 cells.
to the results on the MCF-7 cell line 10 and 11 were
the most active compounds. In a concentration of 5
mM 10 suppressed the cell growth completely (T/C=
3.3. Discussion
The results of this paper demonstrate clearly that
acetylenehexacarbonyldicobalt complexes represent a
novel class of antitumor drugs. Their cytotoxic potency
depends on the structure of the used acetylene ligand.
Among the complexes tested, the hexacarbonyl[2-
propinylacetylsalicylate]dicobalt (10) possesses the
highest antitumor activity. 10 reduced the growth of
H2981 lung adenocarcinoma and 3677 human
melanoma cells [9] as well as the proliferation of human
0%) while 11 showed a maximum effect of T/Ccorr
=
20%.
As a third in vitro test we used the human LNCaP/
FGC prostate cancer cell line. However, complex 10
with the highest cytotoxic potency was only half as
active as cisplatin (see Fig. 7). In a 5 mM concentration
the growth of LNCaP/FGC prostate cancer cells was
reduced to T/Ccorr=48%.

K. Schmidt et al. / Inorganica Chimica Acta 306 (2000) 6–16
15
Fig. 7. Effect of 10 (0.5, 1.0, 5.0 mM) on the proliferation of human LNCaP/FGC cells.
As a first hypothetical mode of action a nucleophilic
attack at the carbonyls must be discussed. In acetylene-
hexacarbonyldicobalt complexes these ligands have a
partial positive charge at the carbon atoms (docu-
mented by the unusually low-field resonances of the
CoꢀCO at l:199) which allows a nucleophilic attack
at these centers. Such reactions are well known in the
literature and were investigated intensively. The group
of Beck could demonstrate a bimolecular reaction of
the SN2-type for the interaction of hexacarbonyl com-
plexes with N-nucleophiles (NH3 or NHR−) [20]. In a
first reaction step the nucleophile binds to CO and a
rearrangement reaction leads to an isocyanato deriva-
tive. Based on MO-calculations they concluded that the
reaction rate depends on the magnitude of the positive
charge at the C-atom.
A second possible center for a nucleophilic attack is
represented by the alkyne ligand. Due to the p-bond the
CꢀC triple bond becomes electron deficient. This leads
e.g. to a deshielding of the C2 carbon atoms as demon-
strated by the low field shift of the resonances in the
13C NMR-spectra. It can be assumed that especially C2
may be subject to a nucleophilic attack.
As a third mode of action, an exchange of the ligands
at the metal atoms by bionucleophiles is conceivable.
For example the CO-ligands could be substituted by
other electron donors such as amine- or R2S-groups.
The results of this study revealed that also steric
effects must be taken into consideration, since the
spectroscopic studies proved comparable electronic en-
vironments at the conceivable reaction centers of the
complexes. Large substituents at C2 or methyl groups at
C3 lower the antitumor activity as documented by the
effects of the complexes 3, 10 and 11.
mammary (MCF-7 and MDA-MB-231) and prostate
carcinoma cells (LNCaP/FGC). On the MCF-7 and
MDA-MB-231 cell line it is more active than cisplatin
(compare Figs. 4–6) while it is only half as active
against LNCaP/FGC cells (compare Fig. 7). The
growth of 3677 human melanoma and H2981 lung
adenocarcinoma cells was influenced by 10 in a 10 times
higher concentration than the phenylenediamine mus-
tard reference substance.
From these results it could be deduced that the
2-propinylsalicylate residue mediated the high activity
for mammary carcinoma cells. The 2-propinylsalicylate
complex 11 as well as the 2-propinylacetylsalicylate
complex 10 exhibit the highest activity against mam-
mary carcinoma cell lines.
To estimate the significance of the binding of the
alkynes to the hexacarbonyldicobalt moiety for the
cytotoxic effects, we tested possible decomposition
products of an oxidative cleavage and subsequent hy-
drolysis of the complexes 10 and 11 under identical
conditions on the MCF-7 cell line (see Fig. 5). As
presented in Fig. 5, the 2-propinylsalicylate (11a), the
2-propinylacetylsalicylate (10a), the salicylic acid (11b)
as well as the acetylsalicylic acid (1) are completely
inactive, although acetylsalicylic acid (1) is known to
have antitumor activity [19]. These results and the
inactivity of CoCl2 and Co2(CO)8 (see Fig. 3) indicate
clearly that the antitumor properties are mediated by
the intact acetylenehexacarbonyldicobalt complexes.
The mode of action, however, is unknown. But we
assume an interaction of the acetylenehexacarbonyldi-
cobalt moiety with biological targets, since substitution
reactions at the bound ligands are possible.

16
K. Schmidt et al. / Inorganica Chimica Acta 306 (2000) 6–16
pound, Ge-132, in: J.D. Nelson, C. Grassi (Eds.), Current
4. Conclusions
Chemotherapy and Infectious Disease, The American Society for
Microbiology, Washington DC, 1980.
Although their mode of action is unknown,
acetylenehexacarbonyldicobalt complexes represent a
new class of antitumor active metal complexes. Hexa-
carbonyl[2-propinylacetylsalicylate]dicobalt (10) was
found to be the most active derivative among the new
complexes with high activity against mammary car-
cinoma cell lines.
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M. Jung thanks Professor Dr B. Unterhalt, Mu¨nster,
for his general support. Thanks are due to the Fonds
der Chemischen Industrie and ViaPharm GmbH, Haan
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