Platinum(IV) complexes featuring one or two axial ferrocene bearing ligands - Synthesis, characterization, and cytotoxicity

Article abstract of DOI:10.1002/ejic.201301282

Ferrocenyl compounds show interesting antiproliferative properties. Consequently, ferrocene bearing moieties were prepared and coupled for the first time to anticancer platinum(IV) complexes. The compounds, featuring either one or two axially coordinated ferrocene-containing ligands, were fully characterized by ESI-MS and multinuclear (¹H, ¹³C, ¹⁵N, and ¹⁹⁵Pt) one- and two-dimensional NMR spectroscopy. Their cytotoxicity was investigated in three human cancer cell lines deriving from ovarian carcinoma (CH1), colon carcinoma (SW480), and non-small-cell lung carcinoma (A549) by means of the colorimetric MTT assay. Promising IC ₅₀ values in the low micromolar range in CH1 and SW480 human cancer cells were found. Copyright

Full text of DOI:10.1002/ejic.201301282

FULL PAPER
DOI:10.1002/ejic.201301282
Platinum(IV) Complexes Featuring One or Two Axial
Ferrocene Bearing Ligands – Synthesis, Characterization,
and Cytotoxicity
Jelena Banfic´,[a] Anton A. Legin,[a] Michael A. Jakupec,[a]
Markus Galanski,*[a] and Bernhard K. Keppler*[a,b]
Keywords: Platinum / Ferrocene / Antitumor agents / Cytotoxicity / Metallocenes
Ferrocenyl compounds show interesting antiproliferative
properties. Consequently, ferrocene bearing moieties were
prepared and coupled for the first time to anticancer plati-
num(IV) complexes. The compounds, featuring either one or
two axially coordinated ferrocene-containing ligands, were
fully characterized by ESI-MS and multinuclear (1H, 13C,
15N, and 195Pt) one- and two-dimensional NMR spectroscopy.
Their cytotoxicity was investigated in three human cancer
cell lines deriving from ovarian carcinoma (CH1), colon carci-
noma (SW480), and non-small-cell lung carcinoma (A549) by
means of the colorimetric MTT assay. Promising IC50 values
in the low micromolar range in CH1 and SW480 human can-
cer cells were found.
pounds has recently received more and more interest. Plati-
num(IV) agents are kinetically inert, thus opening up the
possibility of being administered orally and thereby en-
abling chemotherapy to become a more convenient treat-
ment. Furthermore, six instead of only four coordination
sites may be varied, facilitating a better fine-tuning of the
pharmacological properties of the drug. By now, four plati-
num(IV) compounds have found their way into clinical tri-
als, namely tetraplatin, iproplatin, satraplatin, and LA-12.[4]
Tetraplatin was abandoned due to its high toxicity in
vivo, whereas iproplatin showed a lack of activity on ac-
count of its slow reduction rate.[5] Satraplatin failed to gain
approval by the FDA but is still being investigated in com-
bination therapy, while its derivative, LA-12 was in phase I
clinical trials after having shown good in vivo results in
mice.[4] Besides platinum-based metallodrugs, transition-
metal-based complexes, such as ferrocene-bearing com-
pounds, have shown strong antiproliferative effects in vitro
as well as antitumor effects in vivo.[6,7] The most intensively
investigated ferrocene-based compounds with anticancer
potential are ferrocifens, prepared by replacing a phenyl
group with ferrocene in tamoxifen, a selective estrogen re-
ceptor modulator (SERM) used in hormone-dependent
(ER+) breast cancer treatment. In 1996, Jaouen et al. cou-
pled ferrocene to 4-hydroxytamoxifen and modified the
length of the carbon chain in order to optimize its pharma-
cological properties (Figure 2).[8] The hydroxyferrocifens
show antiproliferative activity in hormone-dependent and
in hormone-independent cells, whereas tamoxifen and hy-
droxytamoxifen are inactive in the latter.[9] Investigation of
ferrocene-based anticancer agents ranged from design of
ferrocenium salts (first investigated by Köpf-Maier et al. in
Introduction
In 1965, Barnett Rosenberg discovered accidentally the
ability of cisplatin to inhibit cell proliferation.[1] After cis-
platin had been granted approval in 1978 as a chemothera-
peutic,[2] many efforts have been made to develop novel
platinum compounds with anti-proliferative potential. Be-
sides cisplatin, two so-called second- and third-generation
platinum drugs, namely carboplatin and oxaliplatin, gained
worldwide approval as antineoplastic drugs (Figure 1).
Three other platinum-based compounds, nedaplatin, loba-
platin, and heptaplatin, gained regional approval in Japan,
China, and South Korea, respectively.[3] Due to severe side-
effects of cisplatin and its analogues and tumor resistance,
and due to the fact that platinum(II) compounds are kinet-
ically labile agents, the investigation of platinum(IV) com-
Figure 1. Worldwide approved platinum-based drugs: cisplatin
(left), carboplatin (middle), and oxaliplatin (right).
[a] Institute of Inorganic Chemistry, University of Vienna,
Waehringer Strasse 42, 1090 Vienna, Austria
E-mail: markus.galanski@univie.ac.at
bernhard.keppler@univie.ac.at
http://anorg-chemie.univie.ac.at
[b] Research Platform “Translational Cancer Therapy Research”,
University of Vienna,
Waehringer Strasse 42, 1090 Vienna, Austria
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1984)[10] over combination of ferrocenyl groups with transi- according to literature methods (Scheme 1).[15] The acid was
tion-metal complexes and to coupling of ferrocene moieties converted into the acyl chloride, subsequently brought to
to drug delivery systems, such as polymers and nanopar- reaction with the desired diamine, N-boc-ethylenediamine,
ticles.[9] During the past two decades, the synthesis of sev- N-boc-ethanolamine or amino alcohol, and characterized
eral platinum(II) compounds bearing ferrocene moieties has by 1H NMR spectroscopy. Precursors L2, L4, and L6 could
been accomplished.[9,11–13] Some of these compounds have directly be used without further purification for coupling
shown promising IC50 values (Figure 3): (i) Nieto et al. re- with compounds 2, 8, and 10 (Schemes 2 and 3), respec-
cently published the successful synthesis and first cell cul- tively. Before amidation, the N-boc protecting groups were
ture tests of a cis-configured ferrocene-platinum derivative, removed from precursors of L3 and L5 by reaction with
which showed in vitro activity in human breast, cervix, lung conc. trifluoroacetic acid (TFA).
and colon cancer cells in the low micromolar range (par-
All investigated complexes were prepared from K2[PtCl4]
tially exceeding the antiproliferative activity of cisplatin);[11] according to literature methods by conversion into two dif-
(ii) a trans-dichloridoplatinum(II) complex featuring two ferent platinum(II) precursors, dichlorido(ethane-1,2-di-
ferrocene-bound phosphane units was designed by Schulz amine) platinum(II) and (1R,2R-diaminocyclohexane)(oxal-
et al., which yielded low IC50 values in the micromolar ato)platinum(II). Both complexes were oxidized sub-
range in the ovarian cancer cell line A2780.[13] A plati- sequently in the presence of hydrogen peroxide to yield di-
num(IV)-ferrocenyl system was described recently.[14] How- hydroxidoplatinum(IV) complexes 1 and 7 (Schemes 2 and
ever, with the aim of combining the advantages of antican- 3). Preparation of dicarboxylic acids 2 and 8 was performed
cer platinum(IV) complexes with the therapeutic potential with succinic anhydride. Activation of the uncoordinated
of ferrocene-based compounds, we report here the synthe- carboxylic acid groups followed by conversion into amides
sis, characterization, and antiproliferative potential of the was accomplished by 1,1Ј-carbonyldiimidazole (CDI). The
first anticancer platinum(IV) complexes bearing ferrocene formed platinum(IV) imidazolides were brought to reaction
moieties as axial ligands.
in situ with ferrocene-bearing amines L2, L3, and L5, which
resulted in amides 4, 3, and 11, respectively. Synthesis of
esters 5 and 12 was carried out by using the peptide cou-
pling reagent N,NЈ-dicyclohexylcarbodiimide (DCC) and 4-
(dimethylamino)pyridine. Since complexes 3, 4, 5, 11, and
12 show only poor solubility in water, analogous complexes
with only one axial ferrocene fragment were prepared. For
that purpose, (1R,2R-diaminocyclohexane)oxalatoplati-
num(II) was oxidized with peracetic acid in acetic acid,
which yielded acetato(1R,2R-diaminocyclohexane)hydrox-
ido(oxalato)platinum(IV) (9). Thus, carboxylation with suc-
Figure 2. Molecular structures of SERM tamoxifen (left) and hy- cinic anhydride of only one axial coordination site was fac-
droxyferrocifens (right).
ile, which resulted in an enhanced solubility in water of the
substance when converted into esters and amides, respec-
tively. Esterification and amidation were achieved by the
same reaction pathways as described above. The final prod-
ucts 13, 14, and 15 were isolated in moderate yields of 20–
41%.
Spectroscopic Characterization
The final products 3–5 and 11–15 were fully charac-
terized by elemental analysis, ESI-MS and multinuclear
(1H, 13C, 15N, 195Pt) 1D and 2D NMR spectroscopy,
whereas structures of ferrocene precursors L2–L6 were veri-
1
fied by H NMR spectroscopy. Carboxylation and further
derivatization can best be judged by NMR spectroscopy.
Figure 3. Cytotoxic platinum(II) complexes with ferrocene-bearing
By analysis of the 2D NMR spectra, all protons of the
ligands.
final products could be assigned except for the two methyl-
ene groups in the axial position nearest to the platinum nu-
cleus because of several overlaps. It was possible to distin-
guish between the three quaternary C=O carbon atoms be-
cause of the long-range shift correlation signals arising
Results and Discussion
Synthesis
For generation of ferrocene precursors L2–L6, the syn- from the carbon atoms and protons of the cyclopentyl ring
thesis started with preparation of ferrocenecarboxylic acid or protons belonging to the residue of the coupled ligand.
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Scheme 1. Synthesis of ferrocene precursors L2–L6 and NMR numbering scheme.
Scheme 2. Synthesis of complexes 3–5 and NMR numbering scheme.
486
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Scheme 3. Synthesis of platinum(IV) complexes 11–15 and NMR numbering scheme.
As described in the literature,[16] novel PtIV complexes with Cytotoxicity
the Cl2N2O2 coordination sphere show 195Pt resonances in
the region around 2656 ppm, whereas resonances for oxali-
The cytotoxicity of dichlorido(ethane-1,2-diamine)plati-
platin-type platinum(IV) complexes appear in the region of num(IV) complexes 3–5, as well as of the oxaliplatin deriva-
3230 ppm. As anticipated, the 195Pt resonances are compar- tives 11–15, was determined in cisplatin-sensitive CH1
able for oxaliplatin-based PtIV complexes 13, 14, and 15 (ovarian carcinoma) and in intrinsically cisplatin-resistant
with one acetato and one derivatized carboxylato ligand in SW480 (colon carcinoma) as well as in A549 (non-small-
the axial position to those for their biscarboxylated ana- cell lung carcinoma) cancer cells by means of the MTT col-
logues. ESI-MS spectra were measured in the positive as orimetric assay (Table 1). In general, the antiproliferative
well as in the negative mode. The highest peak observed in potency of novel ferrocene-containing complexes is highest
each spectrum in the positive mode is [M + Na]+, which is in the ovarian carcinoma cell line CH1 with promising IC50
also in accordance with the calculated values.
values in the low micromolar range (0.84–2.3 μm). Remark-
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ably, the equatorial ligand sphere and the number of ferro- suitability according to the prodrug concept cannot be as-
cene moieties of the platinum compounds seem to have no sessed based on these in vitro investigations alone. Further
pronounced influence on these properties. In contrast, the experiments, especially in vivo, are warranted.
cytotoxicity of all tested complexes in the lung carcinoma
Exemplarily, one of the ferrocene compounds, namely
cell line A549 is only moderate with IC50 values well above L6, was tested for its antiproliferative potential. IC50 values
20 μm. Antiproliferative potency in SW480 colon carcinoma were mostly not reached with maximum tested concentra-
cells was observed in an intermediate range, and a clear- tions of 320 μm, attesting a very low cytotoxicity. The plati-
cut structure–activity relationship in this cell line could be num(IV) precursor 8 shows also a low antiproliferative ac-
drawn. Compounds 3–5 featuring more cisplatin-like equa- tivity (Table 1), however, when L6 and 8 were brought to
torial ligands, i.e. ethane-1,2-diamine and dichlorido, were reaction resulting in ester 12, a significant increase in cyto-
less cytotoxic (IC50 = 32–46 μm) than oxaliplatin-type com- toxicity (one order of magnitude as compared to 8) was
plexes 11–15 (IC50 = 2.7–5.6 μm).
observed, warranting further studies.
Table 1. Cytotoxicity of complexes 3–5 and 11–15 in CH1, A549,
and SW480 cancer cells.
Conclusions
IC50 [μM][a]
Platinum(IV) complexes featuring axially coordinated
ferrocene-bearing ligands were synthesized for the first time
and fully characterized by multinuclear NMR spectroscopy.
Taking into account that platinum(IV) complexes act as
prodrugs, which have to be activated in the organism, they
show promising IC50 values in the low micromolar range in
CH1 and SW480 human cancer cells.
Compound
CH1
SW480
A549
3
1.5Ϯ0.4
1.6Ϯ0.4
2.3Ϯ0.5
0.86Ϯ0.20
1.2Ϯ0.1
0.84Ϯ0.13
1.6Ϯ0.1
1.3Ϯ0.0
0.14Ϯ0.03
0.18Ϯ0.01
55Ϯ28
43Ϯ5
32Ϯ11
46Ϯ9
69Ϯ15
65Ϯ8
84Ϯ1
Ͼ50
38Ϯ4
24Ϯ6
48Ϯ2
49Ϯ12
1.3Ϯ0.4
0.98Ϯ0.21
–
4
5
11
4.4Ϯ1.4
3.9Ϯ1.3
2.7Ϯ1.0
4.8Ϯ1.3
5.6Ϯ0.9
3.3Ϯ0.4
0.29Ϯ0.05
44Ϯ9
12
13
14
15
cisplatin[b]
oxaliplatin
8[c]
Experimental Section
K2[PtCl4] was obtained from Johnson Matthey (Switzerland), fer-
rocenecarboxylic acid from Alfa Aesar. All other chemicals were
purchased from Aldrich, Fluka, Acros, or Fisher and used without
further purification. When platinum(II) complexes were involved,
syntheses were carried out under light protection; glass-coated
magnetic stirring bars were used for each synthesis. For reactions
in water, doubly distilled osmosis water was used.
L6
Ͼ280
Ͼ320
Ͼ320
[a] 50% inhibitory concentrations in the MTT assay (96 h expo-
sure). Values are means (Ϯ standard deviations) obtained from at
least three independent experiments. [b] Data taken from ref.[17] [c]
Data taken from ref.[18]
.
The latter behavior was expected, since oxaliplatin is ap-
proved for clinical treatment of metastatic colorectal carci-
noma and shows above-average in vitro activity in many
intrinsically cisplatin-resistant colon carcinoma cell lines
such as SW480. As could be shown by evaluating monoace-
tato complexes 13, 14, and 15, the linking fragment between
the carboxylatoplatinum(IV) moiety and the ferrocene part
(NH–CH2–CH2–NH vs. NH–CH2–CH2–O vs. O–CH2–
CH2–NH) has nearly no influence on cytotoxicity. Further-
more, no substantial differences in the antiproliferative be-
havior of bis- and mono(ferrocene) derivatives (11 vs. 14
and 12 vs. 15) could be observed.
There is also no difference depending on the length of
the linking methylene moiety (NH–CH2–CH2–NH vs. NH–
CH2–CH2–CH2–NH) when comparing the cytotoxicity of
compounds 3 and 4. Generally, all complexes are less active
by factors of 6–16 in the cisplatin-sensitive cell line CH1
than cisplatin. Likewise, oxaliplatin derivatives 11–15 are by
an order of magnitude less potent than oxaliplatin in
SW480 cells. All novel complexes presented in this manu-
script are hence less effective than cis- and oxaliplatin, but
it should be kept in mind that cis- and oxaliplatin are plati-
num(II) complexes, whereas the central platinum ion in
complexes 3–5 and 11–15 has the oxidation state +4. This
means that they act as prodrugs and, consequently, have to
Starting compounds L1–L6 and precursors,[19–22] (SP-4–2)-(trans-
1R,2R-diaminocyclohexane)oxalatoplatinum(II) (6), (OC-6–33)-
bis(3-carboxypropanoato)dichlorido(ethane-1,2-diamine)plati-
num(IV) (2), (OC-6–33)-bis(3-carboxypropanoato)(trans-1R,2R-di-
aminocyclohexane)oxalatoplatinum(IV) (8), 9, and 10,[23–26] were
synthesized according to literature methods.
Physical Measurements: Elemental Analysis was carried out by the
Microanalytical Laboratory of the University of Vienna with a
2400 CHN Elemental Analyzer manufactured by Perkin–Elmer.
For each submitted substance, the percentage of the elements car-
bon, hydrogen, and nitrogen was determined. Electrospray ioniza-
tion mass spectra were recorded with a Bruker Esquire3000 ion
trap spectrometer with MeOH as solvent, the molecular mass was
determined in the positive as well as in the negative mode. NMR
spectra were recorded on a Bruker FT-NMR Avance III 500 MHz
instrument at 500.32 (1H), 125.81 (13C), 50.70 (15N), and 107.55
(
195Pt) MHz. 2D NMR measurements were recorded by using stan-
dard pulse programs. Chemical shifts were referenced relative to
the solvent signal for 1H and 13C spectra; for 15N and 195Pt spectra,
the external standards 15NH4Cl and K2[PtCl4] were used, respec-
tively.
Synthesis of Ferrocene Precursors
Ferrocenyl Chloride (L1): Ferrocenecarboxylic acid was dissolved
in abs. CH2Cl2 (typically 700 mg in 5 mL of abs. CH2Cl2) under an
argon atmosphere, and oxalyl chloride (2 equiv.) was added. The
solution was stirred for 20 min at room temperature, and the sol-
be activated in the organism by reduction. Therefore, their vent was removed under reduced pressure. The obtained dark red
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substance was dissolved in CH2Cl2 and used without purification
for subsequent reactions.
(550.4 μL, 9.12 mmol) and triethylamine (847 μL 6.04 mmol) in
abs. CH2Cl2 under an argon atmosphere and left to stir for 1 h.
The solution was filtered and extracted three times with water. The
organic phase was dried with MgSO4, and the solvent was removed
N-3-(Aminopropyl)ferrocenecarboxamide (L2): Compound L1
(3.04 mmol) was dissolved in abs. CH2Cl2 (5 mL) and added slowly
over 2 h at –23 °C to a solution of 1,3-diaminopropane (2.5 mL,
30.4 mmol) in abs. CH2Cl2 (50 mL). The solution was filtered (in
order to get rid of the dimer) and extracted three times with water;
the organic phase was dried with MgSO4. After removal of the
solvent under reduced pressure, the product was suspended in di-
ethyl ether and filtered through a G-4 glass filter. The product was
dried in vacuo. Yield: 235 mg, 33%. 1H NMR (CDCl3): δ = 6.82
(br. s, 1 H, NH), 4.69 (t, 3J = 1.9 Hz, 2 H, H3), 4.35 (t, 3J = 1.9 Hz,
2 H, H4), 4.22 (s, 5 H, H1), 3.53 (m, 2 H, CH2), 2.94 (m, 2 H,
CH2), 1.76 (m, 2 H, CH2) ppm.
1
under reduced pressure. Yield: 380 mg, 50%. H NMR (CDCl3): δ
3
3
= 6.15 (br. s, 1 H, NH), 4.71 (t, J = 1.5 Hz, 2 H, H3), 4.40 (t, J
= 1.6 Hz, 2 H, H4), 4.25 (s, 5 H, H1), 3.84 (t, 3J = 4.9 Hz, 2 H,
CH2), 3.59 (m, 2 H, CH2) ppm.
Synthesis of precursors 9 and 10
(OC-6–44)-Acetato(1R,2R-diaminocyclohexane)hydroxido(oxal-
ato)platinum(IV) (9): Compound 6 (3 g, 7.6 mmol) was suspended
in acetic acid (60 mL), peracetic acid (3.3 mL, 2.5 equiv.) was
added, and the solution stirred for 30 min. The solution turned
clear and was left to stir for 24 h. Acetic acid was removed under
reduced pressure; the residue was dissolved in acetic acid (20 mL)
and evaporated again. The product was suspended in ethyl acetate,
methanol was added until the appearance of the suspension did
not change any more, and the suspension was filtered. Solvents
were removed on a rotary evaporator, and the white solid was dried
in vacuo. Yield: 2.43 g, 66%. 1H NMR ([D6]DMSO) = 8.59 (br. m,
1 H, NH2), 8.18 (br. m, 1 H, NH2), 7.80 (br. m, 1 H, NH2), 7.10
(m, 1 H, NH2), ≈2.55 (H1/H2, underneath the DMSO peak), 2.07
(m, 2 H, H3/4/5/6), 1.92 (s, 3 H, H10), 1.54–1.29 (br. m, 4 H, H3/
4/5/6); 1.13 (m, 2 H, H3/4/5/6) ppm.
N-2-(Aminoethyl)ferrocenecarboxamide (L3): Compound L1
(3.48 mmol) was dissolved in abs. CH2Cl2 (6 mL) and then added
via a Teflon cannula to a stirred solution of N-boc-ethylenediamine
(826 μL, 5.22 mmol) and triethylamine (964.7 μL 6.96 mmol) in
abs. CH2Cl2 under an argon atmosphere and left to stir for 1 h.
The solution was extracted three times with water, the organic
phase was dried with MgSO4, and the solvent was removed under
reduced pressure. Yield: 512 mg, 33% of the N-boc-protected prod-
uct. 1H NMR (CDCl3): δ = 6.30 (br. s, 1 H, NH), 4.67 (t, 3J =
3
1.8 Hz, 2 H, H3), 4.31 (t, J = 1.9 Hz, 2 H, H4), 4.18 (s, 5 H, H1),
3.42 (m, 2 H, CH2), 2.90 (m, 2 H, CH2), 1.63 (s, 9 H, H10) ppm.
The product was dissolved in trifluoroacetic acid (4 mL) and left
to stir for 5 min. Subsequently, trifluoroacetic acid was removed
under reduced pressure, and the crude product was extracted three
times with CH2Cl2 and with a saturated NaHCO3 solution. The
organic phase was dried again over MgSO4, and the solvent was
removed under reduced pressure.
(OC-6–44)-Acetato(3-carboxypropanoato)(1R,2R-diaminocyclo-
hexane)oxalatoplatinum(IV) (10): Compound 9 (1 g, 2.04 mmol)
and succinic anhydride (306.2 mg, 3.06 mmol) were suspended in
abs. DMF (15 mL) and stirred for 24 h at 50 °C. The solvent was
removed under reduced pressure, and the crude product was sus-
pended in acetone. Methanol was added stepwise until the amount
of insoluble solid did not change any more. The solution was fil-
tered, the solvents were removed under reduced pressure, the resi-
due was dissolved in acetone again, and ethyl acetate was added
until no further precipitation was observed. The product was fil-
tered, washed with diethyl ether, and dried in vacuo. Yield: 945 mg,
79%. 1H NMR ([D6]DMSO): δ = 12.13 (br. m, 1 H, OH), 8.11–
8.52 (br. m, 4 H, NH2), 2.58 (m, H1/H2), 2.40 (m, 2 H, H12/H13),
2.12 (m, 2 H, H3/H4/H5/H6), 1.97 (s, 3 H, H10), 1.51 (m, 2 H, H3/
H4/H5/H6), 1.41 (m, 2 H, H3/H4/H5/H6), 1.17 (m, 2 H, H3/H4/
H5/H6) ppm.
(S)-N-(1-Hydroxypropan-2-yl)ferrocenecarboxamide (L4): Com-
pound L1 (2.17 mmol) was dissolved in abs. CH2Cl2 (4 mL) and
then added via a teflon cannula to a stirred solution of (S)-2-
amino-1-propanol (606 μL, 4.34 mmol) and triethylamine
(604.9 μL 4.34 mmol) in abs. CH2Cl2 under an argon atmosphere
and left to stir for 1 h. The solution was extracted three times with
water, and the organic phase dried with MgSO4. The solvent was
removed under reduced pressure. Yield: 171 mg, 28%. 1H NMR
(CDCl3): δ = 5.83 (m, 1 H, NH), 4.72 (m, 1 H, H3), 4.69 (m, 1 H,
H3), 4.39 (s, 2 H, H4), 4.25 (s, 5 H, H1), 3.79 (m, 1 H, H6/H7),
3
General Procedure for the Synthesis of Complexes 3, 4, 11, 13, and
14: 1,1Ј-Carbonyldiimidazole and the prevailing complex (2, 8 or
10) were dissolved in abs. DMF stirred for 10 min at 60 °C and
simultaneously flushed with argon. The solution was cooled down
to room temperature, the required amine was added (L2, L3 or
L5), and the solution was left to stir for 24 h. The solvent was
removed under reduced pressure; the crude product was dissolved
in acetone and precipitated with diethyl ether. The product was
filtered and subsequently purified by column chromatography.
3.65 (m, 1 H, H6/H7), 2.96 (m, 1 H, H6/H7), 1.30 (d, J = 6.9 Hz,
3 H, H8) ppm.
[(2-Aminoethoxy)carbonyl]ferrocene
(L5):
Compound
L1
(3.04 mmol) was dissolved in abs. CH2Cl2 (6 mL) and then added
via a teflon cannula to a stirred solution of N-boc-ethylenediamine
(705.4 μL, 4.56 mmol) and triethylamine (847 μL 6.04 mmol) in
abs. CH2Cl2 under an argon atmosphere and left to stir for 1 h.
The solution was extracted three times with water, and the organic
phase dried over MgSO4. The solvent was removed under reduced
pressure. Yield: 384 mg, 32.5% of the N-boc-protected product. 1H
NMR (CDCl3): δ = 4.85 (t, 3J = 1.8 Hz, 2 H, H3), 4.45 (t, 3J =
1.8 Hz, 2 H, H4), 4.32 (m, 2 H, CH2), 4.24 (s, 5 H, H1), 3.52 (m,
2 H, CH2), 1.48 (s, 9 H, H10) ppm. The product was dissolved in
trifluoroacetic acid (4 mL) and left to stir for 5 min. Subsequently,
trifluoroacetic acid was removed under reduced pressure, and the
crude product was extracted three times with CH2Cl2 and with a
saturated NAHCO3 solution. The organic phase was dried again
over MgSO4, and the solvent removed under reduced pressure.
General Procedure for the Synthesis of Complexes 5, 12, and 15: To
a stirred solution of 4-dimethylaminopyridine, carboxylic acid (2,
8 or 10), and the alcohol (L4 or L6) was added N,NЈ-dicyclohex-
ylcarbodiimide over 100 min. under an argon atmosphere. The
solution was left to stir for 24 h at room temperature. The volume
was reduced to 3 mL, and the suspension was filtered. The remain-
ing solution was evaporated, and the crude product was purified
by column chromatography.
(OC-6–33)-Dichlorido(ethane-1,2-diamine)bis(4-{[2-ferrocenoyl-
amino]ethyl}amino)-4-oxobutanoatoplatin(IV) (3): 1,1Ј-Carbonyl-
diimidazole (92 mg, 0.57 mmol), 2 (144 mg, 0.26 mmol) in abs.
DMF (6 mL), L3 (175 mg, 0.64 mmol) in abs. DMF (2 mL). The
{[(2-Hydroxyethyl)amino]carbonyl}ferrocene (L6): Compound L1
(3.04 mmol) was dissolved in abs. CH2Cl2 (6 mL) and then added
via a teflon cannula to a stirred solution of ethanolamine
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crude product was purified by column chromatography (EtOAc/
MeOH, 2:1). The obtained product was dried in vacuo. Yield:
(t, 3J = 5.5 Hz, 2 H, NH), 4.77 (m, 4 H, H17), 4.50 (m, 4 H, H18),
4.23 (s, 10 H, H19), 4.13 (t, J = 5.8 Hz, 2 H, H14), 3.37 (m, 2 H,
3
69 mg, 26%, yellow powder. C36H46Cl2Fe2N6O8Pt·4H2O: calcd. C H13), 2.69 (m, 2 H, H1/H2), ≈2.5 (H10/H11 underneath the DMSO
37.91, H 4.77, N 7.37; found C 37.69, H 4.25, N 7.11. 1H NMR peak), 2.35 (m, 2 H, H10/H11), 2.10 (m, 2 H, H3/H6), 1.50 (m, 2
([D6]DMSO): δ = 8.45 (br. s, 2 H, NH2), 7.94 (br. m, 2 H, NH), H, H4/H5), 1.40 (m, 2 H, H3/H6), 1.18 (m, 2 H, H4/H5) ppm. 13
C
7.83 (br. m, 2 H, NH–COCp), 4.76 (br. m, 4 H, H10), 4.34 (br. m, NMR ([D6]DMSO): δ = 180.7 (C9), 172.0 (C12), 171.1 (C15), 163.8
4 H, H11), 4.16 (s, 10 H, H12), 3.22 (m, 4 H, H7), 3.18 (m, 4 H,
H6), 2.67 (br. m, 4 H, H1), ≈2.5 (H3/H4, underneath the DMSO
peak), 2.32 (t, 3J = 7.3 Hz, 4 H, H3/H4) ppm. 13C NMR ([D6]-
DMSO): δ = 181.7 (C2), 172.0 (C5), 169.6 (C8), 77.0 (C9), 70.4
(C11), 69.9 (C12), 68.6 (C10), 49.2 (C1), 39.3 (C6/7), 39.1 (C6/7),
32.2 (C3/C4), 31.8 (C3/C4) ppm. 15N NMR ([D6]DMSO): δ = 91.4
(NH), 85.8 (NH–COCp), –4.4 (NH2) ppm. 195Pt NMR ([D6]-
DMSO): δ = 2655 ppm. MS (180 °C): m/z = 1091 [M + Na+].
(C7/C8), 71.8 (C18), 71.1 (C16), 70.3 (C17), 70.1 (C17/C19), 62.9
(C14), 61.2 (C1/C2), 38.3 (C13), 31.6 (C3/C6/C10/C11), 31.4 (C3/
C6/C10/C11), 24.0 (C4/C5) ppm. 15N NMR ([D6]DMSO): δ = 88.8
(NH), –6.3 (NH2) ppm. 195Pt NMR ([D6]DMSO): δ = 3233 ppm.
MS (180 °C): m/z = 1164 [M + Na+], 1040 [M – H+].
(OC-6–33)-(trans-1R,2R-Diaminocyclohexane)bis[4-(2-ferrocenoyl-
amino)ethoxy]-4-(oxobutanoato)oxalatoplatin(IV) (12): 4-Dimethyl-
aminopyridine (7.3 mg, 0.06 mmol), N,NЈ-dicyclohexylcarbodiim-
(OC-6–33)-Dichlorido(ethane-1,2-diamine)bis(4-{[3-ferrocenoyl- ide (136.2 mg, 0.66 mmol) in abs. DMF (5 mL), 8 (190.4 mg,
amino]propyl}amino)-4-oxobutanoatoplatin(IV) (4): 1,1Ј-Carbonyl-
diimidazole (109 mg, 0.67 mmol), 2 (184 mg, 0.33 mmol) in abs.
DMF (7 mL), L2 (235 mg, 0.82 mmol) in abs. DMF (2 mL). The
crude product was purified by column chromatography (CHCl3/
0.30 mmol) in abs. DMF (8 mL), L6 (247 mg, 0.90 mmol). The
crude product was purified by column chromatography (CHCl3/
MeOH, 8:1). The obtained product was dried in vacuo. Yield:
67 mg, 20 %, yellow powder. C42H50Fe2N4O10Pt·H2O: calcd. C
MeOH, 5:1). The obtained product was dried in vacuo. Yield: 43.50, H 4.52, N 4.83; found C 43.27, H 4.21, N 4.78. 1H NMR
73 mg, 20%, yellow powder. C38H50Cl2Fe2N6O8Pt·3H2O: calcd. C
([D6]DMSO): δ = 8.35 (br. m, 2 H, NH2), 8.17 (br. m, 2 H, NH2),
3
39.67, H 4.91, N 7.30; found C 39.55, H 4.57, N 7.20. 1H NMR 7.89 (t, J = 5.5 Hz, 2 H, NH), 4.79 (m, 4 H, H17), 4.35 (s, 4 H,
([D6]DMSO): δ = 8.45 (br. s, 2 H, NH2), 7.85 (br. m, 2 H, NH), H18), 4.23 (s, 10 H, H19), 4.12 (t, 3J = 5.7 Hz, 2 H, H13), 3.42 (m,
7.75 (br. m, 2 H, NH–COCp), 4.77 (br. m, 4 H, H11), 4.34 (br. m, 2 H, H14), 2.69 (m, 2 H, H1/H2), ≈2.5 (H10/H11, underneath the
4 H, H12), 4.16 (s, 10 H, H13), 3.18 (m, 4 H, H8), 3.12 (m, 4 H,
DMSO peak), 2.15 (m, 2 H, H10/H11), 2.10 (m, 2 H, H3/H6), 1.50
(m, 2 H, H4/H5), 1.38 (m, 2 H, H3/H6), 1.13 (m, 2 H, H4/H5)
ppm. 13C NMR ([D6]DMSO): δ = 179.9 (C9), 172.8 (C12), 169.8
H6), 2.66 (br. m, 4 H, H1), ≈2.5 (H3/H4, underneath the DMSO
3
peak), 2.31 (t, J = 7.1 Hz, 4 H, H3/H4), 1.61 (m, 4 H, H7) ppm.
13C NMR ([D6]DMSO): δ = 181.7 (C2), 171.7 (C5), 169.3 (C9), (C15), 163.9 (C7/C8), 76.8 (C16), 70.5 (C18), 69.9 (C19), 68.7
77.3 (C10), 70.3 (C12), 69.8 (C13), 68.5 (C11), 49.1 (C1), 36.8 (C6/
(C17), 63.3 (C13), 61.3 (C1/C2), 38.2 (C14), 31.4 (C3/C6), 30.9
C8), 36.8 (C6/C8), 32.3 (C3/C4), 31.8 (C3/C4), 30.1 (C7) ppm. 15
N
(C10/C11), 30.0 (C10/C11), 24.0 (C4/C5) ppm. 15N NMR ([D6]-
NMR ([D6]DMSO): δ = 94.1 (NH), 87.9 (NHCOCp), –4.8 (NH2) DMSO): δ = 82.9 (NH), –6.4 (NH2) ppm. 195Pt NMR ([D6]-
ppm. 195Pt NMR ([D6]DMSO): δ = 2656 ppm. MS (180 °C): m/z
DMSO): δ = 3238 ppm. MS (180 °C): m/z = 1164 [M + Na+].
= 1119 [M + Na+], 1095 [M – H+].
(OC-6–44)-Acetato(trans-1R,2R-diamminocyclohexane)[4-(2-ferro-
cenoylamino)ethylamino]-4-(oxobutanoato)oxalatoplatin(IV) (13):
1,1Ј-Carbonyldiimidazole (63.4 mg, 0.40 mmol), 10 (216.8 mg,
0.36 mmol) in abs. DMF (9 mL), L3 (250 mg, 0.92 mmol) in abs.
(OC-6–33)-Dichlorido(ethane-1,2-diamine)bis(4-{S-2-[ferrocenoyl-
amino]propoxy})-4-oxobutanoatoplatin(IV) (5): 4-Dimethylamino-
pyridine (4.9 mg, 0.04 mmol), N,NЈ-dicyclohexylcarbodiimide
(84.2 mg, 0.41 mmol) in abs. DMF (5 mL), 2 (111.4 mg, DMF (2 mL). The crude product was purified by column
0.20 mmol) in abs. DMF (7 mL), L4 (171.4 mg, 0.60 mmol). The chromatography (CHCl3/MeOH, 8.5:1). The obtained product was
crude product was purified by column chromatography (EtOAc/
MeOH, 2:1). The obtained product was dried in vacuo. Yield:
55 mg, 26%, yellow powder. C38H48Cl2Fe2N4O10Pt·3H2O: calcd. C
d r i e d i n va c u o. Yi e l d : 1 0 0 m g , 3 4 % , y e l l ow p owd e r.
C27H35Fe2N3O11Pt·2H2O: calcd. C 37.55, H 4.67, N 6.49; found C
1
37.34, H 4.27, N 6.30. H NMR ([D6]DMSO): δ = 8.41 (br. m, 1
39.60, H 4.72, N 4.86; found C 39.67, H 4.31, N 4.82. 1H NMR H, NH2), 8.31 (br. m, 1 H, NH2), 8.26 (m, 1 H, NH2), 8.20 (m, 1
([D6]DMSO): δ = 8.46 (br. s, 2 H, NH2), 8.43 (br. s, 2 H, NH2),
7.58 (d, J = 8.3 Hz, 2 H, NH), 4.84 (br. m, 2 H, H11), 4.80 (br.
H, NH2), 7.93 (br. m, 2 H, NH), 7.80 (br. m, 2 H, NH), 4.75 (br.
m, 4 H, H19), 4.35 (br. m, 4 H, H20), 4.15 (s, 10 H, H21), 3.22 (br.
3
m, 2 H, H11), 4.35 (br. m, 4 H, H12), 4.21 (m, 2 H, H7), 4.17 (s, m, 2 H, H15), 3.18 (bm, 2 H, H16), 2.67 (m, 1 H, H1/H2), 2.55
10 H, H13), 4.11 (m, 2 H, H6), 3.95 (m, 2 H, H6), 2.65 (br. s, 4 H, (m, H1/H2), ≈2.5 (H12/H13, underneath the DMSO peak), 2.31
H1), 2.56 (m, 2 H, H3/H4), 2.50 (H3/H4, underneath the DMSO
peak), 1.15 (d, 3J = 6.7 Hz, 6 H, H8) ppm. 13C NMR ([D6]DMSO):
δ = 180.0 (C2), 172.7 (C5), 169.1 (C9), 76.8 (C10), 70.5 (C12), 70.4
(C12), 69.9 (C13), 68.9 (C11), 68.5 (C11), 67.0 (C6), 49.2 (C1), 43.9
(m, 2 H, H12/H13), 2.11 (m, 2 H, H3/H6), 1.96 (s, 3 H, H10), 1.50
(m, 2 H, H4/H5), 1.41 (m,2 H, H3/H6), 1.17 (m, 2 H, H4/H5) ppm.
13C NMR ([D6]DMSO): δ = 180.6 (C11), 179.1 (C9), 172.0 (C14),
169.6 (C17), 163.9 (C7/C8), 163.8 (C7/C8), 77.0 (C18), 70.4 (C20),
(C7), 31.3 (C3/C4), 30.1 (C3/C4), 17.7 (C8) ppm. 15N NMR ([D6]- 69.9 (C21), 68.6 (C19), 61.5 (C1/C2), 61.2 (C1/C2), 39.2 (C15), 39.1
DMSO): δ = 96.3 (NH), –5.1 (NH2) ppm. 195Pt NMR ([D6]-
DMSO): δ = 2658 ppm. MS (180 °C): m/z = 1121 [M + Na+].
(C16), 31.6 (C12/C13), 31.5 (C12/C13), 31.3 (C3/C6), 24.0 (C4/C5),
23.9 (C4/C5), 23.4 (C10) ppm. 15N NMR ([D6]DMSO): δ = 91.3
(NH), 85.4 (NH), –6.3 (NH2) ppm. 195Pt NMR ([D6]DMSO): δ =
3236 ppm. MS (180 °C): m/z = 850 [M + Na+].
(OC-6–33)-(trans-1R,2R-Diaminocyclohexane)bis{4-[2-(ferrocenoyl-
oxy)ethylamino]-4-oxobutanoato}oxalatoplatin(IV) (11): 1,1Ј-Car-
bonyldiimidazole (85.6 mg, 0.53 mmol), 8 (151.8 mg, 0.24 mmol) in (OC-6–44)-Acetato(trans-1R,2R-diaminocyclohexane)[4-(2-ferro-
abs. DMF (6 mL), L5 (234 mg, 0.6 mmol) in abs. DMF (2 mL).
The crude product was purified by column chromatography
(CHCl3/MeOH, 8:1). The obtained product was dried in vacuo.
Yield: 112 mg, 41%, yellow powder. PtC42H50Fe2N4O10: calcd. C
44.18, H 4.41, N 4.91; C 43.86, H 4.11, N 4.74. 1H NMR ([D6]-
cenoyloxy)ethylamino]-4-(oxobutanoato)oxalatoplatin(IV) (14): 1,1Ј-
Carbonyldiimidazole (60.6 mg, 0.37 mmol), 10 (201.1 mg,
0.34 mmol) in abs. DMF (9 mL), L5 (200 mg, 0.51 mmol) in abs.
DMF (2 mL). The crude product was purified by column
chromatography (CHCl3/MeOH, 7:1). The obtained product was
DMSO): δ = 8.36 (br. m, 2 H, NH2), 8.15 (br. m, 2 H, NH2), 8.07 d r i e d i n v a c u o. Yi e l d : 7 8 m g , 2 8 % , y e l l o w p ow d e r.
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C27H36Fe2N4O10Pt·H2O: calcd. C 38.31, H 4.41, N 5.07; found C
38.41, H 4.01, N 4.95. H NMR ([D6]DMSO): δ = 8.42 (br. m, 1
H, NH2), 8.31 (br. m, 1 H, NH2), 8.25 (br. m, 1 H, NH2), 8.24 (br.
100 μL per well (final DMSO concentration 0.5%). After con-
tinuous exposure for 96 h, drug solutions were replaced with
100 μL of a RPMI/MTT mixture (6 parts RPMI 1640 medium sup-
1
3
3
s, 1 H, NH2), 8.07 (t, J = 5.6 Hz, 2 H, NH), 4.77 (t, J = 1.9 Hz, plemented with 10% heat-inactivated fetal bovine serum and 2 mm
3
2 H, H19), 4.50 (t, J = 1.9 Hz, 2 H, H20), 4.23 (s, 5 H, H21), 4.13
(t, 3J = 5.8 Hz, 2 H, H16), 3.37 (m, 2 H, H15), 2.71 (br. s, 1 H,
H1/H2), ≈2.55 (m, H1/H2, underneath the DMSO peak), ≈2.50
(H12/H13, underneath the DMSO peak), 2.35 (m, 2 H, H12/H13),
l-glutamine; 1 part MTT solution in phosphate-buffered saline
[5 mg/mL]). After incubation for 4 h, the medium/MTT mixtures
were removed, and the formazan crystals formed by viable cells
were dissolved in 150 μL of DMSO per well. Optical densities at
2.11 (m, H3/H6), 1.96 (s, 3 H, H10), 1.50 (m, 2 H, H4/H5), 1.41 550 nm were measured with a microplate reader (ELx808 Ab-
(m, 2 H, H3/H6), 1.17 (m, 2 H, H4/H5) ppm. 13C NMR ([D6] sorbance Microplate Reader, Bio-Tek, USA), by using a reference
DMSO): δ = 180.6 (C11), 179.0 (C9), 172.0 (C14), 171.1 (C17), wavelength of 690 nm to correct for unspecific absorption. The
163.9 (C7/C8), 163.8 (C7/C8), 71.9 (C20), 71.1 (C18), 70.3 (C19),
quantities of viable cells were expressed in terms of T/C values by
70.1 (C21), 62.9 (C16), 61.4 (C1/C2), 61.2 (C1/C2), 38.3 (C15), 31.5 comparison to untreated control microcultures, and 50% inhibitory
(C3/C6/C12/C13), 31.4 (C3/C6/C12/C13), 24.1 (C4/C5), 23.9 (C4/ concentrations (IC50) were calculated from concentration-effect
C5), 23.4 (C10) ppm. 15N NMR ([D6]DMSO): δ = 87.6 (NH), –6.6 curves by interpolation. Evaluation is based on means from at least
(NH2) ppm. 195Pt NMR ([D6]DMSO): δ = 3236 ppm. (180 °C): m/z
three independent experiments, each comprising triplicates per con-
centration level.
= 851 [M + Na+], 826 [M – H+].
Stability in DMSO/H2O: Since the complexes had to be dissolved
in DMSO and then diluted in aqueous medium for biological
evaluation, stability under these conditions was addressed by NMR
spectroscopy. In deuterated DMSO, all complexes were stable dur-
ing the acquisition of the data (24–60 h), which is in accord with
previous findings in the case of analogous platinum(IV) complexes.
The stability of a representative complex (compound 3) in a mix-
ture of DMSO/D2O was investigated by 1H NMR spectroscopy.
For this purpose, 0.5 mg of complex 3 was dissolved in 0.5 mL of
DMSO mixed with 0.1 mL of D2O. 1H NMR spectra were mea-
sured from time to time. After sequential intervals of 24, 23.5, 8.5,
and 14 h, 0.1 mL of D2O was added, and the mixtures were investi-
gated in the same way. The last mixture, consisting of 0.5 mL of
DMSO and 0.5 mL of D2O, was investigated for 48 h during which
the signals broadened as a result of crystallization of 3. However,
a change in the NMR spectra could not be observed; in sum, 3
proved to be stable over 118 h under these conditions. In order to
exclude precipitation of a decomposition product, 0.5 mL of the
final suspension was mixed with 0.5 mL of DMSO. The clear solu-
(OC-6–44)-Acetato(trans-1R,2R-diaminocyclohexane)[4-(2-ferro-
cenoylamino)ethoxy]-4-(oxobutanoato)oxalatoplatin(IV) (15): 4-Di-
methylaminopyridine (6.6 mg, 0.05 mmol), N,NЈ-dicyclohexylcar-
bodiimide (61.3 mg, 0.30 mmol) in abs. DMF (5 mL), 10 (160 mg,
0.27 mmol) in abs. DMF (8 mL), L6 (133 mg, 0.49 mmol). The
crude product was purified by column chromatography (CHCl3/
MeOH, 8:1). The obtained product was dried in vacuo. Yield:
67 mg, 30%, yellow powder. C27H35Fe2N3O11Pt·1.5H2O: calcd. C
37.91, H 4.48, N 4.91; found C 37.73, H 4.05, N 4.82. 1H NMR
([D6]DMSO): δ = 8.41 (br. m, 1 H, NH2), 8.33 (br. m, 1 H, NH2),
8.26 (br. m, 1 H, NH2), 8.21 (br. s, 1 H, NH2), 7.89 (t, 3J = 5.7 Hz,
2 H, NH), 4.79 (br. m, 2 H, H19), 4.35 (br. m, 2 H, H20), 4.16 (s,
5 H, H21), 4.11 (m, 2 H, H15), 3.41 (m, 2 H, H16), 2.57 (br. s, 2
H, H1/H2), ≈2.5 (m, H12/H13, underneath the DMSO peak), 2.10
(br. s, 2 H, H3/H6), 1.50 (m, 2 H, H4/H5), 1.40 (m, 2 H, H3/H6),
1.15 (m, 2 H, H4/H5) ppm. 13C NMR ([D6]DMSO): δ = 179.8
(C11), 179.0 (C9), 172.8 (C14), 169.8 (C17), 163.9 (C7/C8), 163.8
(C7/C8), 76.8 (C18), 70.5 (C20), 69.9 (C21), 68.6 (C19), 63.3 (C15),
61.4 (C1/C2), 61.2 (C1/C2), 38.2 (C16), 31.4 (C3/C6), 31.3 (C3/C6),
31.0 (C3/C6/C12/C13), 30.0 (C12/C13), 24.0 (C4/C5), 23.9 (C4/C5),
23.4 (C10) ppm. 15NNMR ([D6]DMSO): δ = 82.7 (NH), –6.7
(NH2) ppm. 195Pt NMR ([D6]DMSO): δ = 3238 ppm. MS (180 °C):
m/z = 851 [M + Na+].
1
tion was investigated by H NMR spectroscopy, which proved the
stability of 3 (significant amounts of hydrolysis products could not
be detected).
Evaluation of Biological Activity
Acknowledgments
Cell Lines and Cultivation Conditions: The cytotoxicity tests were
performed in three cancer cell lines: CH1 (ovarian carcinoma),
SW480 (colon carcinoma), and A549 (non-small cell lung cancer).
Adherent cell monolayer cultures were grown in 75 cm2 culture
flasks (CytoOne, Starlab, UK) in complete medium [i.e. minimal
essential medium (MEM) supplemented with 10% heat-inactivated
fetal bovine serum, 1 mm sodium pyruvate, 4 mm l-glutamine, and
1% nonessential amino acids from 100ϫ ready-to-use stock (all
purchased from Sigma–Aldrich, Austria)]. Cell cultures were incu-
bated at 37 °C in a moist atmosphere containing 5% CO2.
The authors are indebted to the late Lloyd R. Kelland of the CRC
Centre for Cancer Therapeutics, Institute of Cancer Research, Sut-
ton, UK and Brigitte Marian (Institute of Cancer Research, De-
partment of Medicine I, Medical University of Vienna, Austria) for
providing cancer cell lines. Financial support by FWF (grant no.
P20683-N19) and COST is gratefully acknowledged.
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