Polymer conjugates of photoinducible CO-releasing molecules

Article abstract of DOI:10.1002/ejic.201100545

CO-releasing molecules (CORMs) were coordinated to polymeric carrier systems for passive transport to tumour tissue or sites of inflammation, as described by the enhanced permeability and retention (EPR) effect. The developed conjugates consist of an organometallic fac-Mn(CO)₃ fragment, which is bound to a methacrylate or methacrylamide polymer backbone by bis(pyridylmethyl)amine-type ligands. The resulting Mn(CO)₃-polymer conjugates were investigated as photoinducible CO-releasing molecules (photoCORMs). Furthermore, three model complexes [LMn(CO)₃]O ₂SCF₃ containing N,N,N-ligands L = bis(pyridylmethyl)amine (bpma), bis(pyridylmethyl)ethanolamine (bpmea) and bis(pyridylmethyl)-p- vinylbenzylamine (bpmvba) were also investigated. The solid-state structure of the bromide complex [(bpmea)Mn(CO)₃]Br^.(CH ₃)₂CHOH (4·(CH₃)₂CHOH) was determined by X-ray analysis. The complexes and the polymer conjugates show photoCORM activity with polymer molecular weights and size distributions suitable for passive drug delivery.

Full text of DOI:10.1002/ejic.201100545

FULL PAPER
DOI: 10.1002/ejic.201100545
Polymer Conjugates of Photoinducible CO-Releasing Molecules
Nadine E. Brückmann,[a] Michaela Wahl,[a] Guido J. Reiß,[a] Marbod Kohns,[b]
Wim Wätjen,[b] and Peter C. Kunz*[a]
Keywords: Carbon monoxide / Carbonyl ligands / Polymer conjugates / Manganese / EPR effect / Cytotoxicity
CO-releasing molecules (CORMs) were coordinated to poly-
meric carrier systems for passive transport to tumour tissue
containing N,N,N-ligands
(bpma), bis(pyridylmethyl)ethanolamine (bpmea) and bis-
L = bis(pyridylmethyl)amine
or sites of inflammation, as described by the enhanced per- (pyridylmethyl)-p-vinylbenzylamine (bpmvba) were also in-
meability and retention (EPR) effect. The developed conju-
gates consist of an organometallic fac-Mn(CO)3 fragment,
which is bound to a methacrylate or methacrylamide polymer
backbone by bis(pyridylmethyl)amine-type ligands. The re-
sulting Mn(CO)3–polymer conjugates were investigated as
photoinducible CO-releasing molecules (photoCORMs).
Furthermore, three model complexes [LMn(CO)3]O2SCF3
vestigated. The solid-state structure of the bromide complex
[(bpmea)Mn(CO)3]Br.(CH3)2CHOH (4·(CH3)2CHOH) was de-
termined by X-ray analysis. The complexes and the polymer
conjugates show photoCORM activity with polymer molecu-
lar weights and size distributions suitable for passive drug
delivery.
locally restricted tissue volume, for example, in tumour tis-
sue, an overall beneficial cytotoxic effect for the organism
should occur.
Introduction
Carbon monoxide is potentially a very useful therapeutic
as it possesses critical signalling functions, which are rel-
evant in inflammation processes, cellular proliferation,
apoptotic cell death and neural transmission.[1–5] The ma-
jority of endogenously produced CO is as a result of the
oxidation of heme, catalysed by the heme oxygenase (HO)
enzymes. The potential therapeutic effects of CO are widely
recognised in vasodilation, inflammation, hypertension, or-
gan transplantation and cerebral malaria.[6–8] There is a
pharmacological requirement, however, to deliver precise
amounts of CO to specific locations under physiological
conditions, which makes the development of CO-releasing
molecules (CORMs) as benign CO sources highly desir-
able.[9–14]
In the treatment of cancerous diseases, conventional
chemotherapy usually entails a series of injections of highly
toxic drugs, which owe their selectivity for cancer cells to
proliferation rates that are much higher than those in heal-
thy tissue. Conventional chemotherapeutics are small
enough to leave the vascular system by passing through
pores in the blood vessel walls. Therefore, they are distrib-
uted throughout the body, which can lead to increased tox-
icities against healthy tissues that also show enhanced pro-
liferative rates, such as in the bone marrow, gastrointestinal
tract and hair follicles. The resulting severe side effects often
restrict the frequency and size of dosages, much to the detri-
ment of tumour inhibition.[17,18]
Both the cytoprotective and cytotoxic properties of CO
can be exploited for therapeutic purposes.[1,15,16] At low
concentrations the cytoprotective activity is dominant,
whereas at very high systemic concentrations of free CO,
for example, by inhalation of CO, toxicity to the whole or-
ganism occurs due to inference with heme proteins involved
in oxygen transport and storage, as well as respiratory func-
tions. If a high concentration of CO can be achieved in a
The selective toxicity of an anticancer drug can be in-
creased either by increasing the dose of the drug that
reaches the diseased tissue or by decreasing the dose that
reaches healthy tissues. Several approaches for improving
the selective toxicity of anticancer therapeutics are being
pursued at present; one of the most promising is the conju-
gation of anticancer drugs to macromolecular carrier sys-
tems. Conjugated to polymers, drugs are limited to the vas-
cular system and can be transported directly to the area
where the drug is required to take effect. This is possible
because macromolecular systems selectively accumulate in
tumour tissue as described by the enhanced permeability
and retention (EPR) effect.[19–22]
[a] Institut für Anorganische Chemie und Strukturchemie,
Heinrich-Heine-Universität Düsseldorf,
Universitätsstr. 1, 40225 Düsseldorf, Germany
Fax: +49-211-8112287
E-mail: peter.kunz@uni-duesseldorf.de
[b] Institut für Toxikologie, Heinrich-Heine-Universität
Düsseldorf,
The concept of polymer–anticancer conjugates was first
proposed in 1975 by Helmut Ringsdorf. These macromolec-
ular prodrugs consist of a minimum of three components:
Universitätsstr. 1, 40225 Düsseldorf, Germany
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejic.201100545.
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P. C. Kunz et al.
FULL PAPER
the polymeric carrier, the biodegradable polymer-drug
linker and the antitumour agent.[23,24]
The synthesis of MA–PLA–bpmea was done by esterifi-
cation of the hydroxy end group with methacryloyl chloride
The choice of an appropriate polymeric carrier is crucial under basic conditions. The bpmea lactide and triethyl-
for systemic administration and 2-hydroxypropyl methacry- amine were reacted in dichloromethane with a slight excess
lamide (HPMA)-based polymers have drawn special atten- of methacryloyl chloride. After work-up, the product was
tion.[25,26] The linear or branched polymer chain usually precipitated with diethyl ether in yields of close to 90%. It
serves as the structural component of a conjugate. Most is important to note that complete conversion to the meth-
of the clinically tested polymer conjugates have the typical acrylated lactides could only be achieved with short chain
structure consisting of a polymer backbone, a linker and lengths of less than ten repeat units.
the bioactive unit. Modern polymer chemistry is developing
Integration of the bpmea-functionalised macromonomer
increasingly intricate polymer carriers, such as multivalent, into a larger carrier system was realised using HPMA as
branched or graft polymers as well as micelles, dendrimers a co-monomer. A monomer ratio (HPMA to MA–PLA–
and the like. Their potential advantages include a more de- bpmea) of 5:1 was used in the experiments; the macro-
fined chemical composition, tailored surface multivalency monomers had an average number of four repeat units.
and creation of defined 3D architectures.[27–30]
AIBN was used as radical starter and 2-cyanoisopropyl di-
The combination of CO-releasing agents and nano-sized thiobenzoate as the chain transfer agent. The polymer P2
carriers was first used in a micellar system and in function- was isolated in 73% yield (Scheme 2). The molecular weight
alised SiO2 nano-particles.[31,32]
is dependent on the initiator to monomer ratio; an increase
We are interested in metal-based compounds for diag- in the chain length is achieved with a decrease in initiator
nostic and therapeutic applications.[33–38] Herein we present concentration. Molecular weights of approximately 50 kDa
the concept of linking photoinducible CO-releasing mole- translate to a hydrodynamic diameter of approximately
cules (photoCORMs) to HPMA-based copolymers thus 10 nm as determined by gel permeation chromatography
generating nanometer-sized functionalised copolymers (GPC) and dynamic light scattering (DLS).
(“nanoCORMs” or “polyCORMs”), which can be used for
the passive delivery of the CO-releasing metallodrugs to tu-
mour tissue or sites of inflammation.
Results and Discussion
The bis(pyridylmethyl)amine (bpma)-functionalised
HPMA copolymer P1 (Scheme 1) was synthesised accord-
ing to a reported procedure.[38] Instead of the conventional
radical polymerisation conditions, we used RAFT condi-
tions and added bis(thiobenzoyl) disulfide as the chain
transfer agent to obtain polymers with low polydisper-
sity.[39] For the preparation of the HPMA copolymer with
ligand-functionalised biodegradable lactide side chains, we
first prepared the ligand-functionalised polylactide (PLA)–
bpmea [bpmea = bis(pyridylmethyl)ethanolamine] accord-
ing to the procedure of Häfeli.[40] Attaching lactide side
chains to polymer carriers can be achieved by turning them
into macromonomers, introducing polymerisable end
Scheme 2. Preparation of the functionalised HPMA–PLA copoly-
groups. Methacrylate (MA) groups open up the possibility mer P2 and corresponding Mn(CO)3 complex.
of polymerising the lactide with a library of co-monomers
through radical polymerisation. In this way, lactide side
chains can be incorporated into large carrier systems with
additional functionalities and properties.
Polymers carrying bpma-type ligands can easily be lab-
elled with the fac-M(CO)3 cores as has been shown using
Re(CO)3.[34,38] This makes the labelling procedure more
convenient than with, for example, functionalised nano-
particles, which have to be labelled using functionalised
Mn(CO)3 complexes.
The Mn(CO)3 cores were coordinated to the carrier by
the addition of an acetone solution of [Mn(CO)3-
(acetone)3]OTf [prepared from Mn(CO)5Br and silver tri-
flate] to methanolic solutions of P1 or P2. The correspond-
ing Mn(CO)3–polymer conjugates precipitated upon ad-
dition of diethyl ether. The manganese content was deter-
mined by atomic absorption spectroscopy (AAS) [Mn-
Scheme 1. Preparation of the Mn(CO)3 complex of the function-
alised HPMA copolymer P1.
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Polymer Conjugates of Photoinducible CO-Releasing Molecules
(CO)3@P1: 8.9%; Mn(CO)3@P2: 2.8%]. This load is within tionality of the ligand could serve as a donor. Using X-ray
the range also found in the micellar systems of Hubbell or analysis we evidenced that the Mn(CO)3 core was facially
the functionalised SiO2 nanoparticles of Schatzschnei- coordinated by the bpma ligand.
der.[31,32]
Crystals of 4·0.5(CH3)2CHOH were grown from a satu-
For comparison, three molecular Mn(CO)3 complexes 1– rated solution of 4 in hot 2-propanol. The manganese cen-
3 of bpma-type ligands were prepared. The complexes 1–3 tre is in a slightly distorted octahedral coordination envi-
serve as models for the coordination environment of the ronment, with the N,N,N ligand and the three carbonyl li-
Mn(CO)3 moiety in the polymers. All bpma-type ligands gands coordinating facially (Figure 2). The metric param-
form complexes of the composition [LMn(CO)3]OTf [L = eters found in the structure of 4·(CH3)2CHOH are within
bpma, bpmea and bis(pyridyl-methyl)-p-vinylbenzylamine the range of these found in manganese complexes of bpma-
(bpmvba)] when Mn(CO)5Br, AgOTf and the ligand are type ligands and Mn(CO)3 complexes with N,N,N li-
reacted in equimolar quantities in refluxing methanol (Fig- gands.[46,47] To the best of our knowledge, this is the first
ure 1).
example of a single-crystal structure of a bpma ligand in
combination with the Mn(CO)3 core.
Figure 1. Mn(CO)3 complexes 1–3 of bpma-type ligands.
Upon coordination, the methylene protons of the 2-pyr-
1
idylmethyl groups in the H NMR spectra at δ = 4.7 to
4.9 ppm are no longer chemically equivalent due to the Cs
symmetry of complexes 1–3 and form an AB spin system.
The IR spectra of the complexes show the characteristic
bands of the ligands and the two bands of a nearly C3v-
symmetrical Mn(CO)3 fragment, with the doubly degener-
ate E band split (Table 1). This splitting has also been ob-
served in the corresponding Re(CO)3 and MoI(CO)3 com-
plexes of a similar bpma-type ligand.[38,41]
Figure 2. Molecular structure of 4·0.5(CH3)2CHOH. Only one of
the two crystallographic independent Mn(CO)3 complexes of the
asymmetric unit is shown. The co-crystallised solvent molecule, the
bromide counter anions and the H atoms have been omitted for
clarity and displacement ellipsoids are drawn at 50% level. Selected
bond lengths [Å] and angles [°]: Mn1–C1O 1.807(4), Mn1–C3O
1.813(4), Mn1–C2O 1.820(4), Mn1–N1 2.040(3), Mn1–N3
2.047(3), Mn1–N2 2.108(3); C1O–Mn1–C3O 93.61(19), C1O–
Mn1–C2O 86.46(18), C3O–Mn1–C2O 88.72(18), C1O–Mn1–N1
174.33(15), C3O–Mn1–N1 91.13(16), C2O–Mn1–N1 96.77(16),
C1O–Mn1–N3 93.18(15), C3O–Mn1–N3 171.28(16), C2O–Mn1–
N3 97.15(16), C1O–Mn1–N2 94.71(15), C3O–Mn1–N2 92.08(15),
C2O–Mn1–N2 178.54(15), N1–Mn1–N2 82.00(12), N1–Mn1–N3
81.81(12), N3–Mn1–N2 81.92(12).
Table 1. IR bands (ATP), half lives for CO release[a] and moles of
CO released[b] for P1, P2 and complexes 1–3.
Compound
νCO [cm–1]
t1/2 [min]
n(CO)
[c]
Mn(CO)3@P1
Mn(CO)3@P2
1
2
3
2037, 1948, 1934
2036, 1945, 1929
2038, 1949, 1933
2038, 1949, 1937
2038, 1950, 1937
20
20
20
20
20
–
–
[c]
1.59
2.65
1.65
[a] Half life time of CO release after irradiation. [b] Equivalents of
CO released per mole of complex as determined by the myoglobin
assay. [c] Not determined (see text).
CO-Release Study
To study the suitability of the compounds to act as novel
CO-releasing molecules (CORMs), the Mn(CO)3-labelled
copolymers Mn(CO)3@P1/2 as well as the manganese com-
The FAB+ mass spectra of complexes 1–3 exhibit peaks plexes 1–3 were investigated by using the UV/Vis-based
for the ions [LMn(CO)3]+ at the expected m/z values. The myoglobin (Mb) assay.[48,49] Upon irradiation of complexes
IR spectroscopic data of the Mn(CO)3 compounds as well 1–3, the corresponding UV/Vis spectra showed the evol-
as the results from the CO-release study (see below) are ution of the myoglobin–CO adduct, and, in the Q-band re-
summarised in Table 1.
gion (500–600 nm), isosbestic points at 504, 517, 550, 570
For a better understanding of the Mn(CO)3 binding in and 586 nm were observed (Figure 3).
the functionalised polymers, we synthesised and determined
As the Mn(CO)3 load of the Mn(CO)3-labelled polymers
the molecular structure of the model complex [(bpmea)- Mn(CO)3@P1/2 is only 8.9 and 2.8%, respectively, a larger
Mn(CO)3]Br (4), which generated crystals of better quality amount (56.1 and 55.5 mg/5 mL) of polymer was used in
than the triflate congener 2. Bpma-type ligands usually co- the solutions for the myoglobin assay. Photoinduced CO
ordinate facially, but they can also coordinate meridio- release was observed for both polymers, but the solutions
nally.[42–45] Additionally, in complexes 2 and 4 the OH func- turned slightly turbid over the course of the reactions. This
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P. C. Kunz et al.
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ated. The reference compound CORM-2, a molecule, which
spontaneously releases CO when dissolved in DMSO, also
did not show any significant toxic effect on the two cell
lines. Complex 3 shows some cytotoxicity as does Mn-
(CO)3@P2. For these two compounds, the cytotoxicity does
not appear to be due to the CO-releasing effect, as the effect
is observed upon irradiation as well as in the dark (see the
Supporting Information). The IC50 values for Mn-
(CO)3@P2 upon irradiation and in the dark were deter-
mined to be (50Ϯ21) and (41Ϯ6) μg/mL in Hct116 and
(60Ϯ13) and (61Ϯ10) μg/mL in HepG2 cell lines, respec-
tively.
The cytotoxic effect of 3 can be rationalised on the basis
that 3 is a styrene derivative; styrene derivatives are potent
mutagens, and are hepatotoxic and pneumotoxic in mam-
mals.[55,56] More interestingly, the polymer conjugate
Mn(CO)3@P2 shows cytotoxic activity, whereas none of the
bpma-type ligands nor the polymer P2 shows marked toxic-
ity. Similar behaviour has been observed in conjugates of
cymantrene, CpMn(CO)3, with cell-penetrating peptides.
The peptide and the cymantrene compound themselves are
not toxic, however, the conjugates show significant cytoto-
xicity, accompanied by differences in the cellular uptake
and intracellular distribution.[53,54]
Figure 3. UV/Vis spectral changes in the Q-band region of a solu-
tion of reduced horse skeletal muscle myoglobin (75 μm) and 3
(20 μm) in 0.1 m phosphate buffer upon irradiation at 365 nm (t =
0–100 min).
led to offsets in the UV/Vis spectra (see the Supporting In-
formation), which were corrected by applying the method
recently described by Fairlamb et al. (Figure 4).[50]
Conclusion
We prepared two HPMA copolymers with suitable pen-
dant ligands for the coordination of Mn(CO)3-based
CORMs. The polymers consist of either HPMA backbones
with incorporated ligating units or HPMA backbones with
biodegradable oligolacide side chains decorated with ligand
moieties. The size and molecular weight of the HPMA–olig-
olactid copolymers can be designed to (1) suit the EPR ef-
fect for passive accumulation and drug delivery and (2) be
degraded after delivery for renal clearance.
The bpma-type complexes 1–3 are effective CO-releasing
molecules and release about two moles of CO per mole
complex upon irradiation. The same still applies when the
Mn(CO)3 complexes are conjugated to polymers (“poly-
CORMs”). Compounds 1, 2 and Mn(CO)3@P1 do not
show any significant cytotoxicity. Complex 3 and the poly-
mer conjugate Mn(CO)3@P2 show cytotoxic effects with
IC50 values of 50–70 μm and 50–60 μg/mL, respectively.
In this paper, we have demonstrated the use of ligand-
functionalised polymers as carrier systems for Mn(CO)3-
based CORMs. The carrier systems must be chosen care-
fully because a non-toxic carrier in conjugation with a non-
toxic Mn(CO)3 complex may result in a polymer conjugate
with a distinct toxicity.
Figure 4. UV/Vis spectral changes in the Q-band region of a solu-
tion of reduced horse skeletal muscle myoglobin (75 μm) and
Mn(CO)3@P1 (20 μm Mn) in 0.1 m phosphate buffer upon irradia-
tion at 365 nm (t = 0–100 min).
The Mn(CO)3 complexes of bpma-type ligands release
half of the CO load upon irradiation within a period of
20 min. For the calculation of the number of moles of CO
released per mole of complex 1, 2, or 3, we determined the
starting concentration of myoglobin from the absorption
data by using ε560nm = 13.8 mmolL–1 cm–1 and applying
Lambert–Beer’s law.[51] By this method we observe a mean
amount of about two moles of CO released per mole of 1
and 3, and three moles CO per mole of 2.
Cytotoxicity
It is known that the conjugation of non-toxic organome-
tallic species to a non-toxic peptide can still lead to cyto-
toxic conjugates.[52–54] Therefore, we tested the cytotoxicity
of the compounds 1–3, P1, P2 and Mn(CO)3@P1/2 in
Hct116 human colon carcinoma and HepG2 human hepa-
toma cells and compared them to the cytotoxicity of tricar-
Experimental Section
bonyldichloridoruthenium(II)
CORM-2). The compounds 1, 2 and Mn(CO)3@P1 do not
dimer
([Ru2(CO)6Cl4],
General: The ligands bis(pyridylmethyl)amine (bpma), bis(pyridyl-
methyl)ethanolamine (bpmea) and bis(pyridylmethyl)-p-vinyl-
show any significant cytotoxicity in the dark or when irradi- benzylamine (bpmvba) were prepared according published pro-
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Polymer Conjugates of Photoinducible CO-Releasing Molecules
cedures.[38,57,58] All chemicals were used as purchased from Aldrich
and Fluka unless otherwise stated. Reactions carried out under in-
ert conditions were done using standard Schlenk techniques. 1H
NMR spectra were recorded with a Bruker AM 200 and Bruker
DRX 500 spectrometer. The spectra were calibrated against the
residual proton signals of the solvents as internal references. The
ESI mass spectra were recorded with an Ion-Trap-API mass spec-
trometer Finnigan LCQ Deca. MALDI-TOF mass spectra were
recorded with a Bruker Ultraflex TOF mass spectrometer. IR spec-
tra were recorded with a Bruker IFS 66 FT-IR spectrometer. The
manganese content was determined using a Perkin–Elmer atomic
absorption spectrometer 3100. Dynamic light scattering (DLS) ex-
periments were carried out with a Malvern HPPS-ET apparatus at
20 °C. The particle size distribution was derived from a deconvol-
ution of the measured intensity autocorrelation function of the
sample by the general-purpose mode algorithm included in the
DTS software. Each experiment was performed five times to obtain
statistical information.
diethyl ether and dried in vacuo; yield 1.00 g (61%). The ratio of
the two monomers in the copolymer was determined by H NMR
1
spectroscopic analysis by comparing the AUCs of the respective
proton signals. The ratio L to HPMA to lactide was approximately
1:4:4. 1H NMR (CDCl3): δ = 1.22 (m, HPMA-CH3), 1.63 (m, PLA-
CH3), 1.98 (s, 3 H MA-CH3), 2.14 (s, HPMA-CH3), 2.98 (m, 2 H,
NCH2CH2O), 3.98 (d, 4 H, pyCH2), 4.00–4.11 (m, HPMA-CH2,
HPMA-CH), 5.1 (m, 4 H, PLA-CH), 7.46–7.70 (m, 6 H, py), 8.54
(m, 2 H, py). IR (ATP): ν = 2991, 2953, 1723, 1138, 1609, 1259
˜
cm–1.
Synthesis of [LMn(CO)3]OTf Complexes 1–3: In a Schlenk tube,
pentacarbonyl manganese bromide (100 mg, 0.36 mmol) and silver
triflate (93.5 mg, 0.36 mmol) were dissolved in anhydrous acetone
(20 mL). After heating at reflux for 1.5 h, the precipitated silver
bromide was filtered off and the clear yellow solution added to
an acetone solution of the respective ligand (0.36 mmol). Further
heating at reflux for 1.5 h led to formation of the product, which
precipitated after reducing the volume of the reaction solution to
5 mL and adding diethyl ether. After filtration, the solid product
was washed with diethyl ether and dried in vacuo.
MA–PLA–bpmea: A solution of methacryloyl chloride (0.42 g,
4.0 mmol) in dichloromethane was added dropwise to a solution of
PLA–bpmea (1.93 g, 3.6 mmol, average of four lactide units) and
triethylamine (0.41 g, 4.0 mmol) in dichloromethane at 45 °C. The
reaction mixture was stirred at this temperature for 3 h. The volume
was reduced to 10 mL and extracted with hydrochloric acid (10 mL,
0.1 m), brine and bidest. water. The combined aqueous phase was
extracted with dichloromethane and the combined organic phase
dried with Na2SO4. The solvent was removed in vacuo after fil-
tration and the resulting oily residue dried under high vacuum;
yield 1.7 g (78%). 1H NMR (CDCl3): δ = 1.58 (m, PLA-CH3), 2.01
(m, 3 H MA-CH3), 2.95 (m, 2 H, NCH2CH2O), 3.98 (d, 4 H,
pyCH2), 5.07–5.33 (m, PLA-CH), 5.67 (s, 1 H, vinylH-cis), 6.26 (s,
1 H, vinylH-trans), 7.18–7.75 (m, 6 H, py), 8.57 (m, 2 H, py) ppm.
MALDI-TOF: m/z (%): 388 (1250) [1 (dimer) PLA–bpmea], 456
(5050) [1 (dimer) MA–PLA–bpmea], 532 (5660) [3 (tetramer) PLA–
bpmea], 600 (5800) [3 (tetramer) MA–PLA–bpmea], 676 (2260) [5
(hexamer) PLA–bpmea], 744 (1600) [5 (hexamer) MA–PLA–
bpmea], 820 (650) [7 (octamer) PLA–bpmea]. Average of four lac-
tide units.
[(bpma)Mn(CO)3]OTf (1): Yield 79%. 1H NMR (CD3OD): δ =
4.79 (s, 4 H, pyCH2), 7.45 (m, 2 H, pyH2), 7.52 (m, 2 H, pyH4),
7.92 (m, 2 H, pyH3), 9.03 (d, 2 H, pyH1) ppm. 13C{1H} NMR
(CD3OD): δ = 62.9, 124.0, 126.78, 140.8, 153.83, 160.5 ppm. IR
(methanol): ν = 2038, 1949, 1933 cm–1. FAB+: m/z (%) = 338 (22)
˜
[{(pyCH2)2NH}Mn(CO)3]+, 254 (10) [{(pyCH2)2NH}Mn]+, 154
(37) [py(CH2)2NCH2NH2]+, 136 (50) [py(CH2)2NH]+, 107 (33)
[py(CH)2NH]+, 89 (77) [PyC]+ cm–1. C16H13F3MnN3O6S (486.99):
calcd. C 39.44, H 2.69, N 8.62; found C 39.22, H 2.60, N 8.39.
1
[(bpmea)Mn(CO)3]OTf (2a): Yield 77%. H NMR (CD3OD): δ =
3
3
3.98 (t, JHH = 5 Hz, 2 H, CH2CH2OH) 4.12 (t, JHH = 5 Hz, 2 H,
NCH2CH2OH), 4.72–4.89 (m, 4 H, pyCH2), 7.46 (m, 4 H, pyH4,2),
7.94 (m, 2 H, pyH3), 8.98 (m, 2 H, pyH1) ppm. 13C{1H} NMR
(DMSO): δ = 57.7, 67.5, 70.5, 122.9, 125.7, 139.9, 152.4, 161 ppm.
IR (methanol): ν = 2038, 1948, 1937 cm–1. FAB+: m/z (%) = 382
˜
(35) [{(pyCH2)2NCH2CH2OH}Mn(CO)3]+, 298 (15) [{(py-
CH2)2NCH2CH2}Mn]+, 154 (40) [(pyCH2)2NCH3]+, 136 (61)
[py(CH2)2N]+, 77 (88) [py]+. C18H17F3MnN3O7S (531.01): calcd. C
40.67, H 3.23, N 7.91; found C 40.67, H 3.48, N 8.02.
P1: The synthesis described here is an improved procedure com-
pared with the one previously described.[36]
1
[(bpmvba)Mn(CO)3]OTf (3): Yield 82%. H NMR (CD3OD): δ =
HPMA (4.33 g, 30.0 mmol) and bis(pyridiylmethyl)-p-vinylbenz-
ylamine (0.96 g, 3.0 mol) were dissolved in acetone (10 mL) and
AIBN (32.1 mg) and bis(thiobenzoyl) disulfide (85.6 mg) added.
The reaction was stirred for 24 h at 60 °C and concentrated to a
viscous oil. The oil was then poured into a mixture of acetone
and diethyl ether (1:4, 200 mL). The precipitated was collected by
filtration, washed with diethyl ether and dried in vacuo. The ratio
of the two monomers in the copolymer was determined from the
1H NMR spectroscopic analysis by comparing the area under the
curves (AUCs) of the respective proton signals. The ratio of L to
HPMA was approximately 1:9; yield 3.5 g (66%). 1H NMR
(CD3OD): δ = 1.01 (CH2 backbone, HPMA-H1), 1.98 (m, HPMA-
H4), 3.51 (m, HPMA-H3), 3.89 (s, 2 H, NCH2Ph), 3.98 (s, pyCH2),
4.05 (m, HPMA-H2), 7.12–7.87 (m, py), 8.68 (m, py) ppm. IR
4.17 (s, 2 H, PhCH2), 4.94 (m, 4 H, [AB]2-system, pyCH2), 5.39 (d,
3JHH = 12 Hz, 1 H, cis-CH2CH), 5.41 (d, 3JHH = 16 Hz, 1 H, trans-
CH2CH), 6.84 (dd, 1 H, CHCH2), 7.39–7.83 (m, 10 H, pyH3–5, Ph),
8.99 (d, pyH1) ppm. 13C{1H} NMR (CD3OD): δ = 68.2, 73.1,
116.6, 124.2, 126.1 (Ph), 128.1 (Ph), 132.8, 137.7, 140.7, 141.0,
153.8, 162.0 ppm. IR (methanol): ν = 2039, 1950, 1937 cm–1. FAB+:
˜
m/z (%) = 454 (60) {[(pyCH2)2N(Ph(CH2)C2H3]Mn(CO)3}+, 370
(34) {[(pyCH2)2N(Ph(CH2)C2H3]Mn}+, 253 (40) [(pyCH2)2-
NC4H6]+, 154 (21) [py(CH2)NC2H4NH2]+, 136 (35) [py(CH2)2-
NH]+, 107 (28) [py(CH2)N]+, 89 (66) [pyC]+, 77 (75) [py]+.
C25H21F3MnN3O6S·CH2Cl2 (690.03): calcd. C 45.36, H 3.37, N
6.10; found C 44.97, H 3.48, N 6.14.
[(bpmea)Mn(CO)3]Br (4): In a Schlenk tube, bromide (100 mg,
0.36 mmol) in anhydrous acetone (20 mL) was heated at reflux for
1.5 h and the clear yellow solution added to an acetone solution of
the bpmea ligand (88 mg, 0.36 mmol). After further heating at re-
flux for 1.5 h, the product precipitated after reducing the volume
of the reaction solution to 5 mL and adding diethyl ether. After
filtration, the solid product was washed with diethyl ether and dried
in vacuo; yield 133 mg (80%). 1H NMR (CD3OD): δ = 3.98 (t,
(ATP): ν = 2980–2800, 1720, 1597, 1572, 743 cm–1.
˜
P2: AIBN (6.75 mg) and bis(thiobenzoyl) disulfide (18 mg) as the
chain transfer agent were added to a solution of HPMA (0.90 g,
6.3 mmol) and MA–PLA–bpmea (0.75 g, 1.3 mmol) in acetone
(10 mL). The solution was heated at reflux for 1 h and concentrated
in vacuo. The viscous solution was poured into a mixture of ace-
tone and diethyl ether (1:3, 200 mL). The product precipitated as
an off-white solid, which was collected by filtration, washed with
3
3JHH = 5 Hz, 2 H, CH2CH2OH) 4.12 (t, JHH = 5 Hz, 2 H,
NCH2CH2OH), 4.72–4.89 (m, 4 H, pyCH2), 7.46 (m, 4 H, pyH4,2),
Eur. J. Inorg. Chem. 2011, 4571–4577
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjic.org
4575

P. C. Kunz et al.
FULL PAPER
7.94 (m, 2 H, pyH3), 8.98 (m, 2 H, pyH1) ppm. IR (methanol): ν
The assay is based on the ability of viable cells to metabolise yellow
˜
= 2038, 1948, 1937 cm–1.
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT, Sigma, Germany) to violet formazane crystals that can be
detected spectrophotometrically. In brief, cells were plated on 96-
multiwell plates with 10.000 cells/well, allowed to attach for 24 h
and then treated with different concentrations of the substances for
24 h. After this time, the treatment medium was changed and cells
were incubated for 3 h under cell culture conditions with 5 mg/mL
MTT. Then the cells were lysed with DMSO. The concentration of
reduced MTT as marker for cell viability was measured photomet-
rically (560 nm) using a Wallace Victor2 1420 multilabel counter
(Perkin–Elmer). The absorbance of untreated control cells was
taken as 100% viability. All tests were performed in triplicate.
Mn(CO)3-Labelling of Copolymers P1 and P2: In a Schlenk flask,
bromide (50 mg, 0.18 mmol) and silver triflate (49 mg, 0.18 mmol)
were dissolved in anhydrous acetone (15 mL). After heating at re-
flux for 1.5 h, the precipitated silver bromide was filtered off and
a methanolic solution of the respective copolymer was added (P1
56 mg, P2 106 mg) to the clear yellow solution. Further heating at
reflux for 1.5 h led to formation of the product, which precipitated
after reducing the volume of the reaction solution to 30 mL and
adding diethyl ether. After filtration, the solid product was washed
with diethyl ether and dried in vacuo.
1
Mn(CO)3@P1: Yield 294 mg (92%). H NMR (CD3OD): δ = 1.05
Supporting Information (see footnote on the first page of this arti-
cle): Uncorrected data for the myoglobin assay of Mn(CO)3@P1
(Figure ESI1), crystallographic data of 4·0.5C3H7OH (Table ESI1)
and data of the cytotoxicity assays.
(CH2, HPMA-H1), 2.01 (m, HPMA-H4), 3.51 (m, HPMA-H3),
3.89 (s, NCH2Ph), 4.01 (m, HPMA-H2), 7.12–7.90 (m, pyH), 8.68
(m, pyH) ppm. IR (methanol): ν = 2980–2800, 2943, 2037, 1948,
˜
1934, 1695, 1612 cm–1. Mn content (AAS): 8.9%.
1
Mn(CO)3@P2: Yield 270 mg (93%). H NMR (CD3OD): δ = 1.23
Acknowledgments
(m, HPMA-CH3), 1.63 (m, PLA-CH3),1.99 (s, MA-CH3), 2.14 (s,
HPMA-CH3), 2.96 {m, [(pyCH2)2NCH2CH2O]}, 4.00–4.12 (m,
HPMA-CH2, HPMA-CH), 5.15 (m, PLA-CH), 7.11–7.70 (m, py),
We would like to thank Prof. Dr. Helmut Ritter, Institut für
Organische Chemie und Makromolekulare Chemie, Heinrich-
Heine-Universität Düsseldorf, for generous access to the facilities
of the institute.
8.55 (m, py) ppm. IR (ATP): ν = 2991, 2943, 2036, 1954, 1940,
˜
1754, 1609 cm–1. Mn content (AAS): 2.8%.
X-ray Crystallography: Single-crystal X-ray diffraction data of
C37H42Br2Mn2N6O9 (4·0.5C3H7OH) were collected at 100 K on an
Oxford Xcalibur diffractometer equipped with an EOS detector
(Oxford Diffraction, Oxford) with Mo-Kα radiation (λ = 0.71073 Å,
graphite monochromator). The structure was solved by direct
methods after empirical absorption correction using spherical har-
monics, implemented in SCALE3 ABSPACK scaling algorithm.[59]
For the structure solution and refinement, the program package
SHELX97 was used.[60] A Table with the crystallographic data of
4·0.5C3H7OH can be found in the Supporting Information.
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Received: May 26, 2011
Published Online: September 7, 2011
Eur. J. Inorg. Chem. 2011, 4571–4577
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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4577