Cell-specific and nuclear targeting with [M(CO)3]+ (M = 99mTc, Re)-based complexes conjugated to acridine orange and bombesin

Article abstract of DOI:10.1002/chem.200700031

Receptor-specific nuclear targeting requires trifunctional metal complexes. We have synthesized [M(L²-pept)(L¹-acr)(CO)₃] (pept = peptide: acr = acridine-based agent) in which the fac-[M(CO) ₃]⁺ moicty (1st function, M = ^99mTc. Re) couples an acridine-based nuclear-targeting agent (2nd function. L ¹-acr) and the specific cell-receptor-binding peptide bombesin (3rd function, L²-pept). The metalmediated coupling is based on the mixed ligand [2+1] principle. The nuclear targeting agents have been derivatised with an isocyanide group for monodentate (L1) and bombesin (BBN) with a bidentate ligand (L²) for complexation to fac-[M(CO)₃]⁺. For nuclear uptake studies, the model complexes [Re(L²)(L ¹-acr)(CO)₃] (L² = pyridine-2-carhoxylic acid and pyridine2.4-dicarhoxylic acid) were synthesized and structurally characterized. We selected acridine derivatives as nucleartargeting agents, because they are very good nucleus-staining agents and exhibit strong fluorescence. Despite the bulky metal complexes attached to acridine. all [Re(L²)(L¹-acr)(CO)₃] showed high accumulation in the nuclei of PC3 and B16F1 cells, as evidenced by fluorescence microscopy. For radio-pharmaceutical purposes, the ^99mTc analogues have been prepared and radioactivity distribution confirmed the fluorescence results. Coupling of BBN to L² gave the receptor-selective complexes [M(L ²-BBN)(L¹-acr)(CO)₃]. Whereas no internalization was found with B16F1 cells, fluorescence microscopy on PC3 cells bearing the BBN receptor showed high and rapid uptake by receptor-mediated endocytosis into the cytoplasm, but not into the nucleus.

Full text of DOI:10.1002/chem.200700031

FULL PAPER
DOI: 10.1002/chem.200700031
3842
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Chem. Eur. J. 2007, 13, 3842 – 3852

FULL PAPER
Cell-Specific and Nuclear Targeting with [M(CO)3]+ (M=99mTc, Re)-Based
Complexes Conjugated to Acridine Orange and Bombesin
Nikos Agorastos,[a] Lubor Borsig,[b] Anabelle Renard,[c] Philipp Antoni,[a]
Giampietro Viola,[d] Bernhard Spingler,[a] Philipp Kurz,[a] and Roger Alberto*[a]
Abstract: Receptor-specific nuclear tar-
geting requires trifunctional metal
complexes. We have synthesized [M-
complexation to fac-[M(CO)3]+. For
showed high accumulation in the nuclei
of PC3 and B16F1 cells, as evidenced
by fluorescence microscopy. For radio-
pharmaceutical purposes, the 99mTc an-
alogues have been prepared and radio-
activity distribution confirmed the fluo-
rescence results. Coupling of BBN to
L2 gave the receptor-selective com-
nuclear uptake studies, the model com-
plexes [Re(L2)(L1-acr)(CO)3] (L2 =pyr-
R
A
N
(pept=pep-
idine-2-carboxylic acid and pyridine-
2,4-dicarboxylic acid) were synthesized
and structurally characterized. We se-
lected acridine derivatives as nuclear-
targeting agents, because they are very
good nucleus-staining agents and ex-
hibit strong fluorescence. Despite the
bulky metal complexes attached to ac-
tide; acr=acridine-based agent) in
which the fac-[M(CO)3]+ moiety (1st
function, M=99mTc, Re) couples an ac-
ridine-based nuclear-targeting agent
(2nd function, L1-acr) and the specific
cell-receptor-binding peptide bombesin
(3rd function, L2-pept). The metal-
mediated coupling is based on the
mixed ligand [2+1] principle. The nu-
clear targeting agents have been deri-
vatised with an isocyanide group for
monodentate (L1) and bombesin
(BBN) with a bidentate ligand (L2) for
plexes
[M
G
N
Whereas no internalization was found
with B16F1 cells, fluorescence micro-
scopy on PC3 cells bearing the BBN
receptor showed high and rapid uptake
by receptor-mediated endocytosis into
the cytoplasm, but not into the nucleus.
ridine,
all
[Re(L2)(L1-acr)(CO)3]
A
Keywords: bioorganometallics
·
fluorescence · nuclear targeting ·
rhenium · technetium
Introduction
Current radiopharmaceutical research focuses on cell-specif-
ic receptor-targeting agents for the diagnosis or therapy of
cancer and other diseases.[1–3] After receptor binding, subse-
quent internalisation and accumulation of the radioconju-
gates, the radionuclides should not be washed out, thereby
ensuring both therapeutic effect and imaging. Therapeutic
effects from b-emitting radionuclides, such as 131I, 176Lu or
186/188Re, to name only a few, are achieved inside and
amongst the cells by the cross-firing effect of high-energy bꢀ
particles. Essential molecules, such as DNA or intracellular
proteins, are affected by direct hits or by reactive oxygen
species (ROS) generated, for example, through ionisation of
water or oxygen.[4–6] Cell death might occur by apoptosis or
by necrosis. Whereas radioimaging allows for sensitive, non-
invasive in vivo imaging of receptor distribution and biologi-
cal processes at reasonable spatial resolution, it is barely
able to visualize the behaviour of compounds on the cellular
or subcellular level. It would be desirable to follow in vitro
in real time the pathway of a radiolabelled compound in
[a] N. Agorastos, P. Antoni, Dr. B. Spingler, Dr. P. Kurz,
Prof. Dr. R. Alberto
Institute of Inorganic Chemistry
University of Zürich
Winterthurerstrasse 190, 8057 Zurich (Switzerland)
Fax : (+41)44-635-68-03
E-mail: ariel@aci.unizh.ch
[b] Dr. L. Borsig
Institute of Physiology
University of Zürich
Winterthurerstrasse 190, 8057 Zurich (Switzerland)
[c] Dr. A. Renard
Institute of Organic Chemistry
University of Zürich
Winterthurerstrasse 190, 8057 Zurich (Switzerland)
[d] Dr. G. Viola
Department of Pharmaceutical Sciences
University of Padova
via Marzolo 5, 35131 Padova (Italy)
Chem. Eur. J. 2007, 13, 3842 – 3852
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R. Alberto et al.
order to know its ultimate site of accumulation. Fluores-
cence microscopy is a highly suited and powerful tool for
this purpose. Hence, structurally identical fluorescent and
radioactive probes would allow the elucidation of the micro-
scopic track of the compound inside the cell, while imaging
with scintigraphy from outside.[7]
tide and
[
a
tridentate chelator coordinated to the
99mTc(CO)3]+ moiety.[17] Although the proof of concept for
nuclear uptake was given, the syntheses of those conjugates
were lengthy and of little flexibility for drug development
with respect to intercalator- and receptor-targeting vector
variation. Furthermore, it did not easily allow the introduc-
tion of a further cell-specific targeting agent such as a pep-
tide. Acridine derivatives are standard compounds for nucle-
ar staining, because they are strongly fluorescent and rea-
sonably water soluble.[18] They can be derivatised at several
positions, while still keeping their ability to reach the cell
nucleus intact. To facilitate the combination of cell-specific
and nuclear-targeting agents, we employed the mixed ligand
[2+1] approach. One of the active molecules is bound to the
metal through a bidentate and the other one through a
Receptor targeting is the common strategy in radiophar-
macy, but cell-specific targeting of the nucleus is less devel-
oped and essentially a “no-go” area (in radiopharmacy).
Beside purely organic compounds, various metal complexes
have recently been shown to stain cellular nucleus compart-
ments very precisely, at low concentration and with high res-
olution.[8,9] Nuclear targeting is challenging, because cell-
specific targeting and penetration of the nucleus wall have
to be combined. Some amino acid sequences, such as che-
motactic TAT or nuclear localising sequence (NLS) peptides,
have been reported[7,10,11] and are known as carriers into the
nucleus, but their combination with cell-receptor-specific
agents has not been described to our knowledge. Nuclear-
targeting agents in combination with cell-receptor-specific li-
gands would provide a new strategy for radionuclide thera-
py: short-range, high-LET Auger electron emitters (LET=
linear energy transfer) such as 125I, 111In or 99mTc as well as
alpha emitters could be delivered to the nucleus to exert
their therapeutic action directly to DNA or RNA. The dose
deposited very locally is much higher than in the case of
low-LET b-emitters.[12–14]
monodentate ligand. The isocyanide group is a strong mono-
G
dentate ligand and suitable for introduction in the basic acri-
dine leads. Synthetically, we performed the reaction of ethyl-
ene diamine with acridine carboxylic acid 1 to yield com-
pound 2. Formamidation of the terminal amino group in 2
gave compound 3 and subsequent dehydration gave the iso-
cyanide derivative 4. To attach a isocyanide moiety to the
N10 of acridine orange (5), an alkylation with N-(4-bromo-
butyl)formamide was carried out to yield 6 followed again
by dehydration to yield the isocyanide derivative 7. Both
synthetic pathways are shown in Scheme 2.
To assess the possibility of cell-specific nuclear targeting
with metal complexes, we have combined the neuropeptide
bombesin (BBN) and acridine derivatives.[15] Bombesin is
neuropeptide of 14 amino acids, originally isolated from
frog skin, with high affinity for the gastrin-releasing peptide
(GRP) receptor. Acridine-based organic compounds are
highly fluorescent and may act as carriers for pendent mole-
cules into the cell nucleus. The two biological functions are
linked to each other by the [M(CO)3]+ moiety, following the
so-called mixed ligand [2+1] approach.[16] The central com-
plex fragment as the radioactive probe represents the third
function. The concept of these trifunctional metal complexes
for cell-specific nuclear targeting is depicted in Scheme 1.
Scheme 2. Synthesis of the monodentate ligands 4 and 7.
The strategy of conjugating a monodentate isocyanide
ligand can be applied to other nuclear-targeting agents as
well.[19] Thus, if the receptor-targeting molecule (a peptide
for instance) is kept unchanged, the nucleus-targeting func-
tion can be altered without substantially varying the overall
composition of the trifunctional conjugate. The isocyanide
ligands 4 and 7 retained their nucleus-staining properties as
could be seen in fluorescence microscopy studies with PC3
and B16F1 cells. Whereas 4 and 7 are not significantly dif-
ferent from their lead compounds, the coupling of a metal
complex will significantly increase the overall size and physi-
cochemical properties. Thus, it is crucial to evaluate if com-
plexes based on the [Re(CO)3]+ core and bound to 4 and 7
would still be nuclear-targeting agents. The remaining two
coordination sites (in which the receptor-targeting function
will be bound later) must be blocked by a bidentate ligand.
Scheme 1. Schematized representation of a trifunctional radiopharma-
ceutical: A) nuclear-targeting molecule, B) radioactive metal complex for
imaging or therapy, C) specific cell-receptor-targeting molecule.
Results and Discussion
Synthetic procedures: We have recently reported about tri-
functional metal complexes composed of the fluorescent or-
ganic dye pyrene, a nuclear localising sequence (NLS) pep-
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Bioorganometallics
FULL PAPER
Table 1. Crystallographic data for complexes 9 and 10.
We selected 2,4-pyridinedicarboxylic acid (2,4-pydic) for this
purpose. The pyridine nitrogen atom and one of the carbox-
ylato groups coordinate to the metal centre, while the
second carboxylate can be used for covalent binding to a
peptide. In a first step, reaction of 2,4-pydic with [Re-
9
10
formula
Mr
C29H23N4O9Re
757.71
C32H34N5O11Re
850.84
183(2)
T [K]
l [Š]
183(2)
0.71073
0.71073
A
N
crystal system
space group
a [Š]
b [Š]
c [Š]
triclinic
P1
triclinic
P1
pydic)(CO)3] (8) in quantitative yield. In a second step li-
gands 4 or 7 were reacted with 8 to yield the final model
complexes [Re(4)(2,4-pydic)(CO)3] (9) and [Re(7)(2,4-
¯
¯
8.0021(11)
13.6225(15)
13.7436(19)
82.058(15)
76.173(16)
85.371(15)
1439.03(3)
2
7.7329(13)
15.175(3)
16.851(3)
71.09(2)
84.845(19)
77.94(5)
1828.9(5)
2
pydic)(CO)3] (10) in good yields (Scheme 3).
A
a [8]
b [8]
g [8]
V [Š3]
Z
1calcd [Mgmꢀ3
]
1.749
4.283
1.545
3.384
m [mmꢀ1
]
max 2q [8]
unique reflns
G.o.fit on F2
R[a,b]
24.0
4297
0.801
0.0655
25.95
6667
0.738
0.0778
0.1769
Scheme 3. Synthesis of compound 10 from [Re
(OH2)3(CO)3]+ (7).
G
wR2[a,c]
0.1381
[a] Observation criterion: [I>2s(I)]. [b] R=ꢀjjFo jꢀjFc jj/ꢀjjFo j.
The X-ray structures of complexes 9 and 10 could be elu-
cidated. ORTEP presentations are given in Figures 1 and 2
and important crystallographic data in Table 1.
[c] wR2={ꢀ[w(F2oꢀF2c)2]/ꢀ[w(F2o)2]}1/2
.
Figure 1. Molecular structure of complex 9. Solvent and hydrogen atoms
are omitted for clarity. Selected bond lengths [Š] and angles [8] for 9 are:
ꢀ
ꢀ
ꢀ
Re1 C11 1.901(19), Re1 N1 2.162(12), Re1 O1 2.117(10); C11-Re1-O1
95.5(7), C11-Re1-N1 171.4(8), O1-Re1-N1 76.2(5).
The X-ray crystal structure analysis of 9 shows strong in-
tramolecular interactions between the two aromatic systems
acridine and pyridine. The relative orientation of these aro-
matic p systems reveals that they are almost parallel to each
other with a minimal interplanar separation of about 3.43 Š.
The intramolecular p stacking of the aromatic rings is strong
and defines the molecular structure, rather than the overall
molecular properties being important for intermolecular p–
p interactions.[20] Presumably, this intramolecular p stacking
contributes to the steric hindrance of the complete complex,
thereby preventing the acridine moiety from intercalating
into the DNA base pairs (see below). In addition complex 9
is negatively charged under physiological conditions, which
further disfavours interaction with the DNA due to the
DNAꢁs negatively charged phosphate backbone. However,
p-stacking interactions could not be confirmed in solution
by NOESY NMR experiments.
Figure 2. Molecular structure of complex 10. Solvent molecules and hy-
drogen atoms are omitted for clarity. Selected bond lengths [Š] and
ꢀ
ꢀ
ꢀ
angles [8]for 10 are: Re1 C11 2.080(15), Re1 N1 2.264(19), Re1 O5
ꢀ
ꢀ
ꢀ
1.88(3), Re1 C3 1.494(16), Re1 C1 1.885(15), Re1 C2 1.915(15); C111-
Re1-O5 85.0(6), C11-Re1-N1 90.5(5), O5-Re1-N1 73.9(8).
from the rest of the complex. Although the solid-state struc-
ture does not necessarily mirror the behaviour of 10 in solu-
tion, the absence of intramolecular p stacking implies that
the compound should be a stronger intercalator (or DNA
binder) than 9, an assumption that could be confirmed by
linear dichroism (LD) measurements as described below.
The acridinium orange unit in 10 forms a layer of p-stacked
aromatic rings together with symmetry-generated molecules.
The following hydrogen bridges could be identified:
O5···O10 2.84 Š (O5 from a bound carboxylate, oxygen co-
ordinating to Re and O10 (x,y,z) is from a water molecule);
In contrast to 9, the crystal structure of complex 10
(Figure 2) shows the acridine orange moiety rotated away
Chem. Eur. J. 2007, 13, 3842 – 3852
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R. Alberto et al.
O6···O11 2.81 Š (O6 is a free carboxylate oxygen atom and
O11 (x,yꢀ1,z) is from a water molecule); O7···O12 2.72 Š
(O7 is a free carboxylate oxygen atom and O12 (ꢀxꢀ1,ꢀy+
1,ꢀz) is from a water molecule); O7···O13 2.66 Š (O7 is a
free carboxylate oxygen atom and O13 (x+1,yꢀ1,z) is from
a water molecule).
orange has an affinity constant of 1.18”105 mꢀ1 and the
parent compound acridine one of 4.50”105 mꢀ1.[22] Both mol-
ecules show high and fast accumulation in the cell nucleus.
Their real-time uptake has been studied by fluorescence mi-
croscopy.[23,24] As a base for nuclear uptake studies, it is of
key importance to know whether 9 and 10 are still penetrat-
ing the cell wall to reach the nucleus. Considering the fact
that bulky metal complexes are attached to these nuclear-
targeting agents, one might expect that this ability is might
be hampered or lost. It is also essential to determine the af-
finity and binding mode of complexes 9 and 10 to DNA in
order to assess a tight interaction that is required for imag-
ing or therapy. We determined the affinity constants by fluo-
rescence titration of the ligands 4 and 7 by calf thymus
DNA (ct DNA) and with complexes 9 and 10, respectively.
We did not find any fluorescence enhancement or absorp-
tion maximum shift with 4 or 9, indicating that these com-
pounds are not binding in an intercalative way. On the other
hand, ligand 7 and complex 10 showed a strong fluorescence
increase and red shifts of the emission maxima upon addi-
tion of ct DNA, indicating intercalation as has been ob-
The bidentate ligand 2,4-pydic can be coupled to the re-
ceptor-targeting peptide bombesin (BBN) by standard pep-
tide synthetic methods directly on a solid-phase support.
BBN binds to the GRP receptor, which is over-expressed on
many tumour cell lines and therefore relevant for cancer
imaging and therapy. The peptide has been extensively stud-
ied for labelling with 111In or 99mTc.[21] Bombesin was syn-
thesised by using standard solid-phase technology. In the
last step prior to full deprotection and cleavage from the
solid phase, the 2-monomethyl ester of 2,4-pydic was cou-
pled to the BBN N terminus by using BOP activation
(BOP=(benzotriazol-1-yloxy)tris(dimethylamino)phospho-
nium hexafluorophosphate). Deprotection and cleavage
from the solid phase, followed by HPLC purification gave
the final product BBN–(2,4-pydic) (11) in good yield.
ESI-MS analysis confirmed the authenticity of the product
m/z 961.51 [M]+. In a next step, 11 was reacted with [Re-
served earlier for
a
PtII complex bound to acridine
orange.[18]
(OH2)3(CO)3]+ in water to get 12 in 92% yield. ESI-MS
Figure 3 shows the dependence of the relative emission in-
tensities of the complex 10 as a function of the concentra-
gave the correct mass of m/z 1230.93 [MꢀOH2]+. The final
step to compound 13 consisted in the introduction of the nu-
clear targeting moiety 7. Reaction of 12 with 7 in water gave
compound 13 (Scheme 4). ESI-MS showed the correct mass
Figure 3. Fluorescence enhancement upon addition of ct DNA in buffer
to a solution of complex 10.
Scheme 4. Derivatisation of bombesin with 2,4-pydic, complexation with
[Re
(OH2)3(CO)3]+ and introduction of the nuclear-targeting agent to
yield the trifunctional compound 13.
A
tion of DNA in a phosphate-buffered solution at pH 7.4.
The concentration of 10 was kept constant. Upon addition
of ct DNA, the emission intensity increased steadily and was
enhanced about threefold at a saturation [DNA]/[Re] ratio
of 20:1. The increase in luminescence intensities of 10, to-
gether with the hypochromic shift observed in the electronic
absorption spectra (Dl=4 nm), are clear indicators for the
intercalation of the acridine moiety of rhenium complex 10
into the DNA double helix.[25,26] Isothermal titration calo-
rimetry (ITC) measurements allows the very sensitive deter-
mination of affinity constants Ki by titration of 7 and 10 into
an aqueous solution of ct DNA. The heat flow measured by
of 1578.76 and infrared spectroscopy evidenced the vibra-
tion frequencies of the carbonyl ligands n(CO) at 1904 and
2023 cmꢀ1, respectively. Compound 13 represents a first ex-
ample of a model trifunctional complex that can also be pre-
pared with 99mTc by an analogous pathway. In complex 13,
the nuclear-targeting molecule 7, bound through the isocya-
nide to the rhenium centre, the receptor-targeting biomole-
cule BBN and the metal centre are combined into a single
molecular entity.
ITC is in direct correlation with the free energy (DGoverall
)
Binding constants and binding mode: Acridine-based organ-
ic dyes are known to be strong DNA binders. Acridine
released by the intercalation of 7 and 10, respectively. The
first affinity constant obtained for ligand 7 was Ki =2.13”
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Chem. Eur. J. 2007, 13, 3842 – 3852

Bioorganometallics
FULL PAPER
105 ꢁ0.43”105 mꢀ1 (DH1 =ꢀ13.0ꢁ0.4 kcalmolꢀ1), which is
more accurate than the values obtained from fluorescence
spectra. For the rhenium complex 10, Ki =1.13”105 ꢁ0.07”
105 mꢀ1 (DH1 =ꢀ8.9ꢁ0.2 kcalmolꢀ1) is smaller than for
ligand 7 as expected. Nevertheless, the values indicate a
high affinity of 10 for ct DNA although the bulky metal res-
idue attached to the targeting agent affects the intercalation
by about a factor of two.
The situation is different for 7 and 10. The LD signals of
7 were negative at all dye/DNA mixing ratios, both in the
UV region at which the DNA and the dye absorb, as well as
for wavelengths at which only the dye absorbs (Figure 5,
top). The LD signals in the long-wavelength absorption re-
The determination of Ki is not sufficient on its own to
identify a particular mode of interaction. Linear dichroism
(LD) measurements are more conclusive, because this meth-
odology has been shown to be an efficient tool to evaluate
the distinct binding mode between a dye and nucleic
acids.[27] Thus, we recorded the absorption and LD spectra
in the presence of ct DNA. The corresponding spectra are
depicted in Figure 4 for the ligand 4 and complex 9.
Figure 4 (top) confirms the intercalation of acridine deriv-
Figure 5. Absorption and LD spectra of ligand 7 (top) and complex 10
(bottom). Spectra were recorded in phosphate buffer 10 mm, pH 7.2, at
different [DNA-bp]/[dye] ratios a) no dye; b) 25; c) 12; d) 5.
G
gions of the dye (400–600 nm) were negative in the presence
of DNA, indicating an induced orientation of the ligand
chromophore upon binding to ct DNA. A significant in-
crease of the LD signal intensities in the absorption band of
the DNA bases (260 nm) was also observed, suggesting that
the ability of the DNA molecules to orient along the flow
lines is increased upon binding to this compound.
A similar result is obtained for compound 10. A small but
significant LD signal can be observed in the dye absorption
region (Figure 5, bottom), although only at the highest dye/
Figure 4. Absorption and LD spectra of ligand 4 (top) and complex 9
(bottom). Spectra were recorded in phosphate buffer 10 mm, pH 7.2, at
ACHTUNG-
different [DNA-bp]/[dye] ratios. a) no dye; b) 25; c) 12; d) 5.
A
[DNA] ratio employed. This suggests the intercalation of 10
into the DNA in agreement with both binding data and fluo-
rescence titration.
ative 4 in ct DNA. It could be expected that complex 9 be-
haves similarly, but fluorescence titrations implied already
that this is not the case. LD spectra confirm this assumption.
Even at a very high dye/DNA ratio no linear dichroism sig-
nals were induced in the absorption region of 9, confirming
the fluorescence titration experiments. Although no evi-
dence from NOESY NMR experiments could be obtained,
the intramolecular p--p stacking as observed in the X-ray
structure is likely to compete for DNA binding.
Cell uptake studies: Due to a lack of reasonable DNA bind-
ing of ligand 4 and complex 9, we focused our in vitro stud-
ies on ligand 7 and complex 10. We selected two different
cell lines for these studies, a B16-F1 mouse melanoma cell
line with no expression of the GRP receptor and a human
prostate adenocarcinoma cell line, PC-3, which does express
the GRP receptor.[28] The latter cell line can be used for tar-
geting studies with the BBN derivative 13. The uptake of
Chem. Eur. J. 2007, 13, 3842 – 3852
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3847

R. Alberto et al.
complex 10 was detected in the green fluorescence filter
(lem =530 nm) as described in the Experimental Section. To
localize the uptake in the cell, we stained nuclei with DAPI
(4’,6-diamidino-2-phenylindol), routinely used in cell biol-
ogy. After exposing the cells to DAPI and 10 (50 mm) for
2 h, both B16-F1 and PC-3 cells were readily loaded with
complex 10 as detected by fluorescence microscopy and de-
picted in Figure 6.
Figure 7. Complex 10 binding to the nuclear DNA of PC-3 cells during
cell division. DAPI staining shows the centrally organized chromosomes
“fingers”, pointing out the early anaphase of the cell cycle. The dominant
complex 10 staining (green) is co-localized with the DAPI staining as
could be appreciated by the light blue colour, indicating co-localization
(overlay). Cells during division have a rounded shape, thus causing a
slight “blurry” image of the surrounding cytoplasm (bar=10 mm).
plex effectively crosses the cell membrane. It is unlikely that
the entrance of this complex is mediated by endocytosis.
Previous findings have shown that the uptake of a europium
complex by cells occurred also at 48C, thereby excluding an
active transport mechanism from this process.[8] To test the
specificity of specific cell targeting with complexes based on
10, the GRP receptor-specific peptide, Bombesin, is conju-
gated in complex 13.
The trifunctional compound 13 was synthesized as out-
lined above. Compound 13 was purified by preparative
HPLC and tested for stability in buffered aqueous solutions.
The complex is stable for at least 24 h and the ligand 7 does
not cleave from the complex. Compound 13 was added to
B16-F1 and PC-3 cells at concentrations of 100 mm and incu-
bated for 2 h. Cells were evaluated by immunofluorescence
microscopy (Figure 8).
Figure 6. Uptake of the complex 10 by B16-F1 and PC-3 cells was evalu-
ated by immunofluorescence microscopy. Cells were incubated with
100 mm complex 10 for 1 h followed by fixation of cells and nuclear stain-
ing by DAPI. A) Overview images of B16-F1 cells showed uptake of 10
in the cytoplasm (green) with a profound accumulation in the cell nuclei
(blue colour), as visualized by a green staining along with DAPI (light
blue colour). PC-3 cells were also loaded with 10 in the cytoplasm
(green), with accumulation in the nuclei. Note the profound concentra-
tion of 10 found in the nucleoli (bar=20 mm). B) High-magnification
images of individual cells, where the localization of 10 in the nucleus is
visible. Single channel images of the cell for DAPI staining (blue) and for
10 staining (green) were co-localized in the merged image (overlay).
Images represent stacks of optical sections from the cell taken over 3 mm
(z section; bar=10 mm).
While there was virtually no uptake of compound 13 in
B16-F1 cells, PC-3 cells were readily loaded with 13 in the
cytoplasm. In contrast to complex 10, the uptake of 13 by
the cells was significantly suppressed in B16-F1 cells, indi-
cating that this peptide-bound metal complex could not
cross the cell membrane anymore. The significant uptake of
compound 13 by PC-3 cells was consistent with the presence
The cationic complex 10 rapidly accumulates in the cell
nuclei, despite of the bulky pendant metal complex
(Figure 6). Both cells, B16-F1 and PC-3, were loaded effi-
ciently with complex 10 as detected by a staining of the cy-
toplasm. The diverse biological background of the cells did
not play a role by the uptake, thus indicating that 10 could
be considered as a nuclear-targeting agent. Interestingly,
complex 10 was accumulated in the nucleoli of PC-3 cells, in
particular. This observation correlated with the earlier find-
ings for europium complex nucleoli targeting.[8] The tight
binding of 10 to DNA was nicely visualized in dividing PC-3
cells (Figure 7). The clear recognition of chromosomes cen-
trally organized in a dividing cell during anaphase co-local-
ized with 10 bound to the DNA (green). The pronounced
accumulation/association of complex 10 with the nuclear
DNA of dividing cells makes this compound of interest for
treatment of cancer, in which the dividing tumour cells are
the primary targets of therapy. However, the proof of princi-
ple for nuclear targeting with 10 requires guarantee for the
cell specificity, which could be addressed through tumour-
specific receptors like GRP.
Figure 8. Cells were incubated with 100 mm of compound 13 for 2 h, fol-
lowed by fixation and nuclear staining with DAPI as described in the Ex-
perimental Section. B16-F1 cells showed very little of the compound as
determined by almost background level green fluorescence in the cyto-
plasm. PC-3 cells were robustly loaded in the cytoplasm by a green fluo-
rescence (bar=20 mm).
Specific cell targeting: The observation that complex 10
readily accumulated in the nucleus indicates that this com-
3848
www.chemeurj.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 3842 – 3852

Bioorganometallics
FULL PAPER
of the GRP receptor by these cells, thus indicating a recep-
tor-mediated active uptake. As can be clearly seen in
Figure 8, no uptake into the nucleus takes place. Cell-specif-
ic nuclear targeting requires cleavage of the peptide from
the metal complex in order to set free the active nuclear-tar-
geting functions. This is feasible by the introduction, for ex-
ample, of pH sensitive cleavable linkers between peptide
and complex. The low pH in the lysosomes would then
induce the release, for example, of complex 10 from 13. Fast
and localized activation of a drug from a prodrug is a well-
known challenge in pharmaceutical chemistry and requires
further investigations in the context of metal complexes.
[
99mTc(7)(11)(CO)3] (18), the homologue of 13. Yields and
reaction conditions are given in Table 2.
Due to the very low concentrations the 99mTc complexes
(ꢂ10ꢀ7 m), common analytical methods such as NMR or
UV/Vis spectroscopy or mass spectrometry cannot be ap-
plied for characterization of the complexes. Usually, charac-
terization is performed by comparing the HPLC traces of
the 99mTc compounds with the fully characterized rhenium
analogues. Equal retention times of the radioactive trace re-
corded with g detection and the UV/Vis trace of the rheni-
um complex indicates identical complexes.
The reaction of [99mTc(OH2)3(CO)3]+ with 2,4-pydic gives
N
14. Retention time of 14 is 19.5min which compares well
with that of the rhenium analogue 8 (18.8 min). Adding
ligand 4 to 14 gives the neutral complex 16, which is more
lipophilic and longer retention times are observed corre-
spondingly. Again, authenticity of 16 is confirmed by com-
paring the retention times for 16 (24.5min) which is equal
to that of the rhenium analogue 9 (23.9 min).
99mTc-labelling studies: The precursor [99mTc(OH2)3(CO)3]+
G
was prepared from [99mTcO4]ꢀ by using the Isolink kit (Mal-
linckrodt Med. B.V. Petten, NL).[29,30] The concentration of
99mTc is in the range of 10ꢀ7 m. After kit preparation, the so-
lution was buffered with 0.1m phosphate buffer to pH 7.4
before further reaction. HPLC analyses with g-detection
were performed to quantify the conversion of the 99mTc. Re-
action time, concentrations and temperature were varied.
Typical labelling yields are given in Table 2.
At a ligand (7) concentration of 10ꢀ4 m, 14 was quantita-
tively labelled at 908C after 40 min to provide the product
17. Compound 17 display a similar HPLC pattern as 13 with
retention times of 23.7 and 23.2 min, respectively.
The reaction of the derivatised bombesin 11 with [99mTc-
Table 2. Yields and reaction conditions for the conversion of
A
[
99mTc
(OH2)(2,4-pydic-
A
[
99mTc(OH2)3(CO)3]+ to [99mTc(L2)(L1)(CO)3]+.
BBN)(CO)3] (15) in good yield after 60 min . The two peaks
at 13.73 and 14.10 min represent the expected diastereomers.
The identity of the peaks was confirmed by comparison with
[Tc(OH2)(CO3)3(L2)]/
[Tc(L1)(CO3)3(L2)]
complex
t
T
[8C]
[L2/L1]
[mm]
Yield
[%]
[min]
2,4-pydic/4
2,4-pydic/7
2,4-pydic-BBN (11)/7
14/16
14/17
15/18
40/60
40
60
75/80
75/90
80/90
0.1
0.1
0.1
>98/95
>98/90
95/95
[Re(OH2)(11)(CO)3]. The introduction of ligand 7 leads to
G
the formation of the positively charged complex 18. While
two peaks are observed in the HPLC trace of complex 15,
corresponding to two diastereomers, only one peak is visible
in the case of 18. This implies that only the thermodynami-
cally more favoured diastereomer is formed or that the two
forms can not be separated under HPLC conditions. The
small peaks at 14.10 and 13.73 min are unreacted 15
(Figure 9).
The [2+1] mixed ligand synthesis of complexes of the gen-
eral type [99mTc(CO)3(L2)(L1)] consists of two consecutive
steps.[16] First, [99mTc(OH2)3(CO)3]+ reacts with a bidentate
C
ligand L2. Then, the monodentate ligand L1 is coordinated
to the sixth available coordination site on the fac-
[
99mTc(CO)3]+ core by replacing the remaining water ligand.
Histidine and cysteine challenge: The stabilities of the la-
belled compounds 16, 17 and 18 were challenged with the
For example, reactions of [99mTc
ligands 2,4-pydic or the BBN conjugate 11 gave [99mTc-
(OH2)(2,4-pydic)(CO)3] (14) and 99mTc-labelled BBN [99mTc-
(OH2)(11)(CO)3] (15), respectively, in near quantitative
N
ACHTREUNG
ACHTREUNG
yield. Reaction conditions are 30–60 min at 708C and L2
concentrations are 10ꢀ4 m. Concentrations of 10ꢀ5 m are pos-
sible without significant decrease of the yield. It should be
noted that no coordination of a second ligand was observed.
In the subsequent step, the monodentate nuclear-targeting
ligands 4 and 7 were introduced. This reaction is significant-
ly slower and higher temperatures are required to bring the
reaction to completion within 60 min. Reaction of 14 with
the acridine derivative 4 gave the radiolabelled complex
[
99mTc(4)(2,4-pydic)(CO)3] (16), and with the acridine
orange compound 7 the cationic complex [99mTc(7)(2,4-py-
dic)(CO)3]+ (17). Complex 17 is the homologue of 10 used
in the previous fluorescence studies. Finally, reaction of 15
Figure 9. Radioactive HPLC trace of 15 (dotted line). Formation of the
final compound 18 (solid line); Linear gradient: ammonium acetate/ace-
tonitrile (90:10) to 100% acetonitrile from 10 to 20 min and then back to
with
7
gave the radioactive trifunctional complex
ammonium acetate/acetonitrile (90:10). The flow rate was 1.5mLmin ꢀ1
.
Chem. Eur. J. 2007, 13, 3842 – 3852
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3849

R. Alberto et al.
ments. To avoid a p–p stacking of the acridine orange residues, a low
concentration of 0.3 mm of 7 and 0.5m m of 10 was chosen. The concen-
tration of the ct DNA was 0.07 mm in the titration with 7 and 0.04 mm for
10. The raw heat–flow versus time data was baseline adjusted, peak inte-
grated, and processed with the Origin Software Package version 7.0 and
the corresponding VP Viewer 2000 ITC to obtain the molar heat flow
per molar ratio. DGoverall, the free energy of each ITC titration, was calcu-
very potent tridentate ligands histidine and cysteine at a
molar ratio of 1/100. The mixtures were kept at 378C, pH 7
for 2, 4 and 12 h. Analysis by HPLC revealed complete sta-
bility of the 99mTc complexes under these conditions. Besides
ꢀ
the intact radiolabelled complexes, no 99mTcO4 could be ob-
served. The stability study of these complexes showed that
no significant ligand exchange and/or decomposition are ex-
pected within 12 h of incubation.
lated from integrated experimental data of the equilibrium constant KITC
.
The sequential binding model fit with three sequential binding sites was
chosen to fit the experimental data.
Cell culture and complex uptake: B16-F1 mouse melanoma cells were
cultured in DMEM supplemented with 10% fetal calf serum. Human
prostate cancer cells, PC-3 were incubated in F12K medium supplement-
ed with 10% fetal calf serum. For microscopy, cells were cultured over-
night on 4-chamber slides (Nunc) in which ꢂ50000 cells were plated.
Next day, cells were washed and fresh medium containing complex 10 or
13 (100 mm) was added. Cells were incubated with the complex for 1–2 h.
After the loading with a complex, cells were washed with phosphate
buffer saline (PBS) and fixed in 3% paraformaldehyde for 10 min in at
RT. After three washings with PBS, cells were incubated in 1 mgmLꢀ1
4’,6-diamidino-2-phenylindole (DAPI) for nuclear staining for 10 min at
RT. Cells were washed three times with phosphate-buffered saline (PBS),
chambers from the slides were detached and cells were covered with cov-
erslips in Prolong Mounting medium (Invitrogen). Slides were evaluated
on Zeiss Axiovert 200m with the corresponding fluorescence filters for
DAPI (lex=360 nm, lem =420 nm) and acridine orange (lex =496 nm,
lem =525 nm). In some cases optical sectioning with Apotom system
(Zeiss) was performed
Conclusion
We have shown the individual steps required for targeting
the nucleus of a specific cell type with rhenium and with
99mTc. A bifunctional nucleus-targeting molecule is able to
carry a metal complex into its target. A trifunctional mole-
cule, which contains, in addition, receptor-specific bombesin,
is only taken up by cells containing the GRP receptor.
These two decisive steps have now to be followed by the
study of trifunctional molecules containing a pH-dependent
cleavable linker between the nucleus-targeting metal com-
plex and the cell-targeting peptide. These studies are cur-
rently underway.
Acridine-4-carboxylic acid (1): The synthesis was performed according to
literature with modifications.[32] Portions (100–200 mg each) of Al foil
(1.7 g, 0.063 mol) were amalgamated with Hg by immersing them for
1 min in a solution of mercuric chloride (6.0 g, 0.022 mol) in ethanol
(50 mL),. After being washed in ethanol, these were added over 20 min
to a refluxing, stirred solution of 9-oxiacridan-4-carboxylic acid (2.0 g,
0.0083 mol) dissolved in 50% aqueous ethanol (15 mL) containing KOH
(530 mg, 0.009 mol). When the reaction was complete, as indicated by
HPLC, 1m aqueous KOH (36 mL) was added, the hot mixture was fil-
tered and the solids were washed with hot 50% aqueous ethanol
(15mL). The combined filtrates were strongly acidified with 12 m HCl,
treated with FeCl3 (500 mg, 0.003 mol), heated under reflux until homo-
Experimental Section
General information: All solvents were of the quality puriss or p.a. The
commercially available reagents were used as received without further
purification. Analytical thin-layer chromatography (TLC) was carried out
by using aluminium-based plates (silica gel 60 F254) from Merck. Plates
were visualized under UV light (l=254 nm). Flash chromatography was
carried out by using Merck silica gel 60 (0.040–0.063 mm) with N2 over-
pressure. Samples were applied as almost saturated solutions in the ap-
propriate solvent. 1H NMR and 13C NMR spectra were performed on a
Varian Gemini 300 and Bruker DRX500 spectrometers at 300 and
75MHz, and 500 and 125MHz respectively. The reported chemical shifts
(in d) are relative to the solvent protons and carbons as a reference. Mul-
tiplicities are reported using the following abbreviations: s (singlet), d
(doublet), t (triplet) and m (multiplet). IR spectra of solids were record-
ed on a Bio-Rad FTS-45spectrometer with samples in compressed KBr-
pellets. Mass spectra were measured on a Merck Hitachi M-8000 spec-
trometer. Linear dichroism (LD) measurements were performed with a
Jasco J500 A spectropolarimeter equipped with an IBM PC and a Jasco J
interface. The sample orientation was produced by a device designed by
Wada and Kozawa for the studies of differential flow dichroism of poly-
mer solution at a shear gradient of 700 rpm in phosphate buffer 10 mm
pH 7.2.[31] High-performance liquid chromatography (HPLC) was per-
formed on a Merck L7000 system by using a Macherey-Nagel EC 250/3
Nucleosil 100–5C18HD column. HPLC solvents were 0.1% trifluoroace-
tic acid as solvent A and methanol as solvent B. The flow rate was
0.5mLmin ꢀ1. Detection was performed at 250 nm. Preparative HPLC
was performed on a Varian Pro Star system by using either a Macherey-
Nagel VP 250/21 Nucleosil 100–7 C18 or a Macherey-Nagel VP 250/40
geneous, and clarified by filtration. Solid K(O2CCH3) was then added to
R
just complete precipitation of the crude product, which was immediately
collected by filtration and washed well with water. It was then dissolved
in dilute aqueous KOH, filtered, diluted while hot with ethanol and then
treated slowly with acetic acid, when acridine-4-carboxylic acid separated
as a pale yellow solid (1120 mg, 61%), suitable for further use. 1H NMR
(500 MHz, [D4]CD3OD): d=9.35(s, 1H), 8.83 (d, 3J
8.47 (d, 3J(H,H)=9.1 Hz, 1H), 8.26–8.23 (t, 3J
(H,H)=8.5Hz, 2H), 8.13
(d, 3J(H,H)=9.0 Hz, 1H), 7.82–7.79 (t, 3J
(H,H)=9.0 Hz, 1H), 7.76–
7.73 ppm (t, 3J(H,H)=8.3 Hz, 1H); 13C NMR (75MHz , [D 4]CD3OD):
A
A
ACHTREUNG
A
ACHTREUNG
AHCTREUNG
d=168.3, 148.5, 144.9, 139.2, 135.7, 134.2, 132.7, 129.6, 129.4, 127.7, 127.5,
126.3, 125.9, 125.2 ppm; IR (KBr): n˜ =1703 (s), 1623 (s), 1569 (s) 1516
(s), 1427 (m), 739 cmꢀ1 (s); HPLC: Rt: 18.4 min; ESI-MS : m/z: 224.00
[M+H]+; elemental analysis (%) calcd for C14H9NO2: C 75.33, H 4.06, N
6.27; found: C 75.72, H 3.99, N 5.82.
Acridine-4-carboxylic acid (2-aminoethyl)amide (2): A suspension of ac-
ridinecarboxylic acid (580 mg, 2.190 mmol) and (benzotriazol-1-yloxy)-
tris(dimethylamino)phosphonium hexafluorophosphate (BOP; 3.48 g,
7.86 mmol) in dichloromethane (25mL) was stirred for 2 h at RT. The re-
action mixture was cooled at 08C, ethylenediamine (1.3 mg, 21 mmol)
was added dropwise and the resulting reaction mixture was stirred for an-
other 3 h at RT. The solvent was removed in vacuo and the crude product
was purified by flash chromatography (silica gel, CH2Cl2/MeOH/
NH4OH-25%, 10:1:0.1, Rf =0.34) to give 2 (326 mg, 56%) as a yellow
Nucleosil 100–7 C18 column with
a
flow rate of 10 mLminꢀ1 and
40 mLminꢀ1. respectively. The solvents were 0.1% trifluoroacetic acid as
solvent A and methanol as solvent B. The gradient used for analysis was
: 0–3 min 100% A, 3.1–9 min 75% A, 9.1–20 min 66% to 0% A, 20–
25min 0% A, 25.1–30 min 100% A. All ITC measurements were carried
1
3
ACHTREUNG
oil. H NMR (500 MHz, [D4]CD3OD): d=8.64 (s, 1H), 8.14 (d, J
8.1 Hz, 1H), 7.90–7.74 (m, 4H), 7.53–7.45 (dd, 3J(H,H)=9.3, 4J
2.4 Hz, 2H), 3.62 (t, 3J
(H,H)=9.6 Hz, 2H), 3.33 (brs, 2H), 2.97 ppm (t,
3J(H,H)=9.6 Hz, 2H); 13C NMR (125MHz, [D 4]CD3OD): d=169.1,
out on
a
MicroCal VP-ITC microcalorimeter at pH 7.0 and 37 (ꢁ
A
ACHTREUNG
0.01)8C. The cell volume was 1.406 mL and the syringe volume had a
maximum capacity of 267 mL. All samples were prepared in a 1 mm
sodium cacodylate buffer at pH 7.0 and degassed before the measure-
AHCTREUNG
AHCTREUNG
3850
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ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 3842 – 3852

Bioorganometallics
FULL PAPER
149.2, 147.3, 139.8, 136.1, 134.7, 133.2, 129.9, 129.4, 128.4, 127.9, 126.3,
2H), 2.04 (brs, 2H), 1.28 ppm (s, 2H); 13C NMR (125MHz,
[D4]CD3OD): d=157.7, 144.7, 144.4, 134.6, 118.8, 115.8, 93.7, 47.7, 42.12,
41.03, 27.6, 23.8 ppm; IR (KBr): n˜ =2145 (w), 1608 (s), 1522 (m), 1504
125.6, 42.1, 37.4 ppm; HPLC: Rt: 19.45min; ESI-MS
: m/z: 265.53
[M+H]+; elemental analysis (%) calcd for C16H15N3O: C 72.43, H 5.70, N
ꢀ1
15.84; found: C 73.82, H 5.65, N 13.22.
(m), 1363 (m), 1165(m), 806 cm (w); HPLC: Rt: 20.7 min; ESI-MS: m/
z: 347.13 [M+H]+; elemental analysis (%) calcd for C22H27N4: C 62.30, H
Acridine-4-carboxylic acid (2-formylaminoethyl)amide (3): Compound 2
(326 mg, 1.23 mmol) was suspended in methyl formate (10 mL). This mix-
ture was refluxed for 3 h, under exclusion of air. After addition of p-tol-
uylsulfonic acid (7 mg) the reaction mixture was refluxed for another
14 h (HPLC-control). The solvent was removed in vacuo and the crude
product purified by flash chromatography (silica gel, CH2Cl2/MeOH/
NH4OH-25%, 10:1:0.1, Rf =0.45) to give 283 mg (79%) of 3 as a yellow
powder. 1H NMR (300 MHz, [D4]CD3OD): d=8.64 (s, 1H), 8.14 (d,
6.42, N 13.20; found: C 61.10, H 6.54, N 12.97.
[Re
A
(8):
(NEt4)2
A
(101 mg,
0.13 mmol) was dissolved in water (2 mL). After addition of AgNO3
(70 mg, 0.4 mmol) and stirring at RT for 3 h, AgBr was removed by filtra-
tion and 2,4-pydic (24 mg, 0.13 mmol) was added to the colourless solu-
tion, which contained [Re
(OH2)3(CO)3]+, followed by stirring at 508C
A
for 2 h. The now yellow solution was cooled down and the product was
allowed to crystallize at 48C for 12 h. The yellow crystals were collected
by filtration, washed with water and dried at high vacuum to give 8
3J
3J
ACHTREUNG
A
G
ACHTREUNG
(brs, 1H), 2.97 ppm (t, 3J
A
(36 mg, 62%). 1H NMR (300 MHz, [D6]DMSO): d=8.96 (d, 3J
ACHTREUNG
[D4]CD3OD): d=168.3, 163.2, 148.5, 139.2, 135.7, 134.2, 132.7, 129.6,
129.4, 127.5, 125.9, 125.2, 43.4, 42.2 ppm; HPLC: Rt: 19.8 min; ESI-MS:
m/z: 293.73 [M+H]+; elemental analysis (%) calcd for C17H15N3O2: C
69.61, H 5.15, N 14.33; found: C 69.29, H 5.25, N 14.55.
9.0 Hz, 1H), 8.34 (d, 3J
A
ACHTREUNG
1H), 7.50 ppm (s, 1H); 13C NMR (75MHz, [D 6] DMSO): d=197.8,
194.4, 174.5, 165.6, 154.5, 153.8, 143.2, 133.2, 128.4 ppm; IR (KBr): n˜ =
2023 (s), 1892 (s), 1717 (w), 1646 (m), 1611 (m), 1374 (w), 1348 (w),
770 cmꢀ1 (w); HPLC: Rt: 17.8 min; ESI-MS: m/z: 437.60 [MꢀOH2]+; ele-
mental analysis (%) calcd for C10H6NO8Re: C 26.45, H 1.33, N 3.07;
found: C 26.15, H 1.67, N 3.07.
Acridine-4-carboxylic acid (2-isocyanoethyl)amide (4): Ethylentriamine
(270 mg, 2.70 mmol) was added to a solution of 3 (258 mg, 0.880 mmol)
in absolute dichlormethane (10 mL). The mixture was cooled to 08C and
phosphorusoxychloride (POCl3; 148.5mg, 0.970 mmol) was added drop-
wise. After 2 h reaction time a solution of sodium carbonate (Na2CO3,
164 mg) in water (1 mL) was added. The reaction mixture was stirred for
1 h at RT. The resulting suspension was then treated with water (10 mL;
till clear) and extracted three times with CHCl3. The organic layer was
dried over Na2SO4 and the solvent was removed in vacuo. The crude
product was purified by flash chromatography (silica gel, CH2Cl2/MeOH,
10:0.5, Rf =0.43) to give 4 (70 mg, 29%) as a yellow powder. 1H NMR
[Re(4)(2,4-pydic)(CO)3] (9): A solution of 8 (29 mg, 0.064 mmol) and 4
(17.5mg, 0.064 mmol) in methanol (2 mL) was stirred at room tempera-
ture for 12 h. HPLC analysis showed complete product formation. The
solvent was removed in vacuo and the crude product was purified by
preparative HPLC to give 9 as yellow powder in a yield of (36 mg, 80%).
Crystals for X-ray diffraction analysis were obtained by slow evaporation
of
a
solution of
9
in methanol/dichloromethane (1:1). 1H NMR
3J
(H,H)=7.1,
(H,H)=6.5Hz, 1H), 8.33 (dd,
(H,H)=1.0 Hz, 1H), 8.26 (d, 3J
(H,H)=8.3 Hz, 1H), 8.20
(H,H)=8.3 Hz, 2H), 7.94 (m, 1H), 7.71 (m, 2H), 7.54 (dd,
(H,H)=6.5, 4J
(H,H)=1.8 Hz, 1H), 4.22 (s, 2H), 3.91 ppm (m, 2H);
(500 MHz, [D4]CD3OD): d=9.15(s, 1H), 8.63 (dd,
ACHTREUNG
(300 MHz, [D4]CD3OD): d=8.64 (s, 1H), 8.14 (d, 3J
7.90–7.74 (m, 4H), 7.53–7.45 (dd, 3J(H,H)=8.7, 4J
(H,H)=2.8 Hz, 2H),
3.62 (t, 3J(H,H)=8.7 Hz, 2H), 2.97 ppm (t, 3J
(H,H)=9.2 Hz, 2H);
A
4J
3J
U
ACHTREUNG
A
N
A
ACHTREUNG
A
N
(d, 3J
ACHTREUNG
13C NMR (75MHz, [D 4]CD3OD): d=168.3, 157.2, 148.5, 139.2, 135.7,
134.2, 132.7, 129.6, 129.4, 127.5, 125.9, 125.2, 44.4, 43.2 ppm; IR (KBr):
n˜ =3053 (w), 2150 (m), 1659 (s), 1624 (m), 1566 (m), 910 (w), 737 cmꢀ1
(m); HPLC: Rt: 21.4 min; ESI-MS: m/z: 275[ M+H]+; elemental analysis
(%) calcd for C17H13N3O: C 74.17, H 4.76, N 15.26; found: C 73.92, H
4.87, N 15.11.
3J
A
ACHTREUNG
13C NMR (125MHz, [D 4]CD3OD): d=193.5, 193.2, 191.0, 174.3, 168.8,
168.1, 163.3, 163.0, 154.3, 151.6, 149.2 148.1, 147.2, 138.4, 136.6, 135.2,
133.3, 130.1, 129.7, 129.0, 128.4, 127.4, 126.3, 119.5, 117.2, 48.6, 39.7 ppm;
IR (KBr): n˜ =2212 (w), 2033 (s), 1946 (s), 1908 (s), 1647 (s), 1381 cmꢀ1
(w); HPLC: Rt: 20.8 min; ESI-MS: m/z: 692.53 [MꢀOH]+.
3,6-Bis-dimethylamino-10-(4-formylaminobutyl)acridinium (6):
A
solu-
[Re(7)(2,4-pydic)(CO)3] (10): A solution of 8 (9 mg, 0.020 mmol) and 7
(7 mg, 0.020 mmol) in methanol/water (2 mL; 1:1) was stirred at 758C for
2 h. HPLC analysis showed complete product formation. The solvent was
removed in vacuo and the product was dried at high vacuum. The crude
product was purified by flash chromatography (basic Alox, CH2Cl2/
MeOH/NH4OH, 5:0.7, 0.05 Rf =0.29). Product 10 (9 mg, 65%) was ob-
tained as an dark orange powder. Crystals for X-ray diffraction analysis
were obtained by slow evaporation of a solution of 10 in methanol/di-
chloromethane (1:1). 1H NMR (700 MHz, [D4]CD3OD): d=8.72 (d,
tion of 5 (250 mg, 0.943 mmol) and 4-bromobutyl formamide (340 mg,
1.886 mmol) in p-xylene (20 mL) was stirred, under N2, for 24 h at 1458C.
The solvent was removed in vacuo and the crude product was purified by
flash chromatography (basic alox, CH2Cl2/MeOH, 5:2, Rf =0.33) to give 6
(179 mg, 52%) as an dark orange powder. 1H NMR (500 MHz,
[D4]CD3OD): d=8.93 (s, 1H), 8.2 (s, 1H), 7.85(d, 3J
7.20 (dd, 3J(H,H)=9.2, 4J
(H,H)=2.1 Hz, 2H), 6.74 (s, 2H), 4.67 (t,
3J
(H,H)=9.3 Hz, 2H),
A
ACHTREUNG
(H,H)=7.7 Hz, 2H), 3.56 (s, 12H), 3.21 (s, 2H), 2.99 ppm (m, 4H);
13C NMR (125MHz, [D 4]CD3OD): d=164.5, 157.4, 144.3, 144.0, 134.4,
134.0, 122.9, 121.1, 115.0, 93.7 (4C), 71.1, 61.9, 41.2 (4C), 40.5, 25.7,
24.2 ppm; IR (KBr): n˜ =1691 cmꢀ1 (s); ESI-MS: m/z: 365.27 [M+H]+.
3J
1H), 7.93–7.88 (m, 1H), 7.29–7.26 (m, 1H), 6.68 (s, 1H), 6.53 (s, 1H),
4.75(t, 3J
(H,H)=7.8 Hz, 2H), 3.95(s, 2H), 3.35(s, 12H), 2.21–2.15(m,
A
AHCTREUNG
2H), 2.04 (brs, 2H), 1.30 ppm (s, 2H); 13C NMR (175MHz,
[D4]CD3OD): d=192.9, 192.7, 190.3, 173.6, 168.5, 163.2, 156.6, 153.5,
150.5, 150.3, 143.8, 143.5, 143.2, 140.1, 133.4, 133.6, 128.5, 126.7, 117.7,
117.5, 114.7, 92.8, 92.4, 47.3, 46.8, 46.0; 44.2, 40.1, 37.5, 27.3, 26.5,
23.5ppm; IR (KBr): n˜ =2212 (w) 2028 (m), 1946 (m), 1907 (m), 1643
(m), 1600 (s), 1503 (m), 1365 (s), 1168 (s), 805 cmꢀ1 (w); HPLC: Rt:
21.8 min; ESI-MS: m/z: 783.43 [M+H]+, 754.87 [MꢀCO]+.
Peptide synthesis: Bombesin (sequence: WAVGHLM) was synthesized
on an Applied Biosystems ABI433 A peptide synthesiser by using stan-
dard Fmoc chemistry and Rink amide MBHA resin (360 mg,
0.66 mmol/g). The following side-chain protected amino acids were used
3,6-Bis-dimethylamino-10-(4-isocyanobutyl)acridinium (7): A mixture of
6 (100 mg, 0.274 mmol) and diisopropylamine (222 mg, 2.2 mmol) in di-
chloromethane (10 mL) was cooled to ꢀ88C and stirred for half hour.
POCl3 (168 mg, 1.1 mmol) was added over a period of 10 min, followed
by stirring at the same temperature for six hours. The reaction mixture
was quenched by cautious addition of an aqueous solution of sodium car-
bonate (20%, 2.3 mL). After stirring at ambient temperature for one
hour, water (2 mL) and dichloromethane (1 mL) were added. The organ-
ic phase was separated and washed with water (3”1 mL). The aqueous
phases were extracted with dichloromethane (3”2 mL) and the com-
bined organic solutions were dried over sodium sulfate. Evaporation of
the solvent gave the crude product as an orange powder. The crude prod-
uct was purified by flash chromatography (basic alox, CH2Cl2/MeOH/
NH4OH-25%, 10:1:0.1, Rf =0.38) to give 7 (51 mg, 54%) as an orange
powder. 1H NMR (500 MHz, [D4]CD3OD): d=8.71 (s, 1H), 7.95(d,
for the synthesis: Fmoc-Ala-OH, Fmoc-Gln
ACHTREUNG
Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Met-OH Fmoc-Trp
A
ACHTREUNG
and Fmoc-Val-OH. Fmoc deprotection was performed by using 20% pi-
peridine in N-methylpirrolidone (NMP). Coupling reactions were carried
out by using 2-(1H-benzotriazole-1-yl)-1,1,3,3,-tetramethylaminium hexa-
fluorophosphate/N-hydroxy-benzotiazole (HBTU/HOBt) (4 equiv) with
3J
6.74 (s, 2H), 4.80 (t, 3J
A
G
A
ACHTREUNG
Chem. Eur. J. 2007, 13, 3842 – 3852
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3851

R. Alberto et al.
diisopropylethylamine (DIEA) (8 equiv). At the end of the synthesis, the
peptide was cleaved from the solid-support and deprotected by shaking
the resin in trifluoroacetic acid/water/triisopropylsilane (95:2.5:2.5
15mL) for 3 h. The resin was removed by filtration, and after concentra-
tion in vacuo, the crude deprotected peptide was precipitated by addition
of cold iPr2O (5mL). After centrifugation, the pellet was washed twice
with iPr2O and dried under high vacuum. The product was then purified
by reverse phase HPLC on a preparative C18 column (Zorbax Eclipse
XDB-C18; 21 mm”250 mm, 7 mm, 80 Š) using a gradient of 5to 50%
[4] C. von Sonntag, M. Dizdaroglu, Carbohydr. Res. 1977, 58, 21–30.
[5] B. Halliwell, J. M. C. Gutteridge, Free Radical Res. 1998, 29, 469–
486.
[6] C. R. Silva, J. O. Valsa, M. S. Canine, A. Caldeira-de-Araujo, M.
Bernardo-Filho, Yale J. Biol. Med. 1998, 71, 7–14.
[7] K. E. Bullok, S. T. Gammon, S. Violini, A. M. Prantner, V. M. Villa-
lobos, V. Sharma, D. Piwnica-Worms, Mol. Imaging 2006, 5, 1–15 .
[8] J. Yu, D. Parker, R. Pal, R. A. Poole, M. J. Cann, J. Am. Chem. Soc.
2006, 128, 2294–2299.
acetonitrile in water (+ 0.1% TFA) over 15min and
a
flow of
[9] A. R. Cowley, J. Davis, J. R. Dilworth, P. S. Donnelly, R. Dobson, A.
Nightingale, J. M. Peach, B. Shore, D. Kerr, L. Seymour, Chem.
Commun. 2005, 845–847.
[10] P. Hꢂfliger, N. Agorastos, B. Spingler, O. Georgiev, G. Viola, R. Al-
berto, ChemBioChem 2005, 6, 414–421.
[11] K. E. Bullok, M. Dyszlewski, J. L. Prior, C. M. Pica, V. Sharma, D.
Piwnica-Worms, Bioconjugate Chem. 2002, 13, 1226–1237.
[12] T. M. Behr, G. Sgouros, V. Vougioukas, S. Memtsoudis, S. Gratz, H.
Schmidberger, R. D. Blumenthal, D. M. Goldenberg, W. Becker, Int.
J. Cancer 1998, 76, 738–748.
[13] G. L. Griffiths, S. V. Govindan, G. Sgouros, G. L. Ong, D. M. Gold-
enberg, M. J. Mattes, Int. J. Cancer 1999, 81, 985–992.
[14] T. M. Behr, M. Behe, M. Lohr, G. Sgouros, C. Angerstein, E. Wehr-
mann, K. Nebendahl, W. Becker, Eur. J. Nucl. Med. 2000, 27, 75 3–
765.
[15] S. R. Nagalla, B. J. Barry, A. M. Falick, B. W. Gibson, J. E. Taylor,
J. Z. Dong, E. R. Spindel, J. Biol. Chem. 1996, 271, 7731–7737.
[16] S. Mundwiler, M. Kündig, K. Ortner, R. Alberto, Dalton Trans.
2004, 1320–1328.
[17] P. Hꢂfliger, N. Agorastos, A. Renard, G. Giambonini-Brugnoli, C.
Marty, R. Alberto, Bioconjugate Chem. 2005, 16, 582–587.
[18] B. E. Bowler, K. J. Ahmed, W. I. Sundquist, L. S. Hollis, E. E.
Whang, S. J. Lippard, J. Am. Chem. Soc. 1989, 111, 1299–1306.
[19] H. J. Pietzsch, A. Gupta, R. Syhre, P. Leibnitz, H. Spies, Bioconju-
gate Chem. 2001, 12, 538–544.
[20] C. A. Hunter, J. K. M. Sanders, J. Am. Chem. Soc. 1990, 112, 5525–
5534.
[21] C. J. Smith, G. L. Sieckman, N. K. Owen, D. L. Hayes, D. G. Mazuru,
R. Kannan, W. A. Volkert, T. J. Hoffman, Cancer Res. 2003, 63,
4082–4088.
15mLmin ꢀ1. HPLC: Rt: 14.5min; ESI/MS: m/z: 812.7 [M+H]+.
Pyridine-2,4-dicarboxylic acid 2-methyl ester bombesin: A solution of
pyridine-2,4-dicarboxylic acid-2-methylester (5.4 mg, 0.0296 mmol) in
DMF (0.5mL) and BOP (26 mg, 0.0591 mmol) was stirred for 3 h at 0 8C.
Then a solution of BBN (18 mg, 0.0197 mmol) and triethylamine (TEA;
18 mg, 0.0597 mmol) in DMF (1 mL) was slowly added. The reaction
mixture was stirred for 3 h at 08C and 1 h at room temperature. The sol-
vent was removed in vacuo and the crude product was purified by flash
chromatography (silica gel, CH2Cl2/MeOH/NH4OH-25%, 5:1:0.1 Rf =
0.08). The product (20 mg, 83%) was obtained as a white powder.
HPLC: Rt: 20.35min; ESI-MS: m/z: 975.83 [M+H]+.
2,4-Pyridine-dicarboxylic acid bombesin (2,4-Pydic–BN; 11): NaOH
(0.1m, 8.0 mg, 0.200 mmol) was added to a solution of pyridine-2,4-dicar-
boxylic acid 2-methyl ester bombesin (20 mg, 0.020 mmol) in DMF
(1 mL). The resulting colourless solution was stirred for 1 h at 08C and
for another hour at room temperature. The solvent was removed in
vacuo and the product was purified by preparative HPLC to give 11
(8 mg, 41%) as a white powder. HPLC: Rt: 19.7 min; ESI-MS: m/z:
961.51 [M+H]+.
[Re(OH2)(11)(CO)3] (12): Compound 7 (1.4 mg, 0.0042 mmol) was
G
added to a solution of 11 (4 mg, 0.0042 mmol) in methanol (2 mL). The
resulting colourless solution was stirred for 1 h at room temperature and
for another hour at 808C. HPLC analysis showed complete product for-
mation. The solvent was removed in vacuo and the product was dried at
high vacuum to give 12 (5mg, 92%) as white powder. HPLC: Rt:
20.3 min; ESI-MS: m/z: 1230.93 [MꢀOH2]+.
[Re(7)(11)(CO)3] (13): Compounds 12 (5mg, 0.0057 mmol) and 7 (2 mg,
0.0057 mmol) in water/methanol (8 mL; 1:1). The orange solution was
stirred for 3 h at 858C. The ESI-MS and HPLC analysis confirmed the
disappearance of 7 and the formation of 13. The solvent was removed in
vacuo and the crude product was purified by semi preparative HPLC to
give 13 (2 mg, 22%) as an orange powder. HPLC: Rt: 23.2 min; IR
(KBr): n˜ =2201 (w), 2023 (m), 1904 (m), 1684 (m), 1601 cmꢀ1 (s); ESI-
[22] M. B. Lyles, I. L. Cameron, Biophys. Chem. 2002, 96, 53–76.
[23] J. K. Frost, H. W. Tyrer, N. J. Pressman, C. D. Albright, M. H. Van-
sickel, G. W. Gill, J. Histochem. Cytochem. 1979, 27, 545–551.
[24] J. F. Golden, S. S. West, J. Histochem. Cytochem. 1974, 22, 495–505.
[25] V. Wing-Wah Yam, K.-W. L. , C. Kung-Kai, R. Yuen-Chong Kong, J.
Chem. Soc. Dalton Trans. 1997, 2067–2072.
[26] F. Pierard, A. Del Guerzo, A. Kirsch-De Mesmaeker, M. Demeu-
nynck, J. Lhomme, Phys. Chem. Chem. Phys. 2001, 3, 2911–2920.
[27] B. Norden, M. Kubista, T. Kurucsev, Q. Rev. Biophys. 1992, 25, 5 1–
170.
MS: m/z: 1578.76 [M+H]+, 1550.61 [MꢀCO]+, 788.84 [M]2+
CCDC-632603 and CCDC-632604 contain the supplementary data for
this paper. These data can be obtained free of charge from The Cam-
bridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_
request/cif.
.
[28] R. La Bella, E. Garcia-Garayoa, M. Bahler, P. Blauenstein, R. Schi-
bli, P. Conrath, D. Tourwe, P. A. Schubiger, Bioconjugate Chem.
2002, 13, 599–604.
[29] R. Alberto, K. Ortner, N. Wheatley, R. Schibli, A. P. Schubiger, J.
Am. Chem. Soc. 2001, 123, 3135–3136.
Acknowledgement
[30] R. Alberto, R. Schibli, A. Egli, A. P. Schubiger, J. Am. Chem. Soc.
1998, 120, 7987–7988.
[31] A. Wada, S. Kozawa, J. Polym. Sci. Part A 1964, 2, 853–864.
[32] G. J. Atwell, G. W. Rewcastle, B. C. Baguley, W. A. Denny, J. Med.
Chem. 1987, 30, 664–669.
This work was supported by Mallinckrodt Med. Inc. Petten NL. We
thank Dr. H. Schmalle for support in one of the X-ray structure analyses.
[1] S. Liu, Chem. Soc. Rev. 2004, 33, 445–461.
[2] S. M. Okarvi, Med. Res. Rev. 2004, 24, 685–686.
[3] P. Blower, Dalton Trans. 2006, 1705–1711.
Received: January 9, 2007
Published online: March 23, 2007
3852
www.chemeurj.org
ꢀ 2007 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2007, 13, 3842 – 3852