Trifunctional 99mTc based radiopharmaceuticals: Metal-mediated conjugation of a peptide with a nucleus targeting intercalator

Article abstract of DOI:10.1039/c0ob00504e

The development of molecular imaging agents with multiple functions has become a major trend in radiopharmaceutical chemistry. We present herein the syntheses of trifunctional compounds, combining an acridine orange (AO) based intercalator with a GRP receptor specific bombesin like peptide (BBN). Metal-mediated conjugation of these two functions via the [2 + 1] approach to the third function, the [M(CO)₃]⁺ (M = ^99mTc, Re) moiety, yielded the final trifunctional molecules. The strongly fluorescent acridine orange, a nuclear targeting agent, has been derivatised with 4-imidazolecarboxylate as a bidentate ligand and bombesin with an isonitrile group as a monodentate ligand. For cell and nuclear uptake studies, [Re(L ¹-BBN)(L²-Ical)(CO)₃] type complexes were synthesized and characterized. For radiopharmaceutical purposes, the ^99mTc analogues have been prepared in a stepwise synthesis. Fluorescence microscopy studies on PC-3 cells, bearing the BBN receptor, showed high and rapid uptake into the cytoplasm. For the bifunctional molecule, lacking the BBN peptide, no internalization was observed.

Full text of DOI:10.1039/c0ob00504e

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PAPER
Trifunctional 99mTc based radiopharmaceuticals: metal-mediated conjugation
of a peptide with a nucleus targeting intercalator†
Karel Zelenka,a Lubor Borsigb and Roger Alberto*a
Received 28th July 2010, Accepted 1st November 2010
DOI: 10.1039/c0ob00504e
The development of molecular imaging agents with multiple functions has become a major trend in
radiopharmaceutical chemistry. We present herein the syntheses of trifunctional compounds,
combining an acridine orange (AO) based intercalator with a GRP receptor specific bombesin like
peptide (BBN). Metal-mediated conjugation of these two functions via the [2 + 1] approach to the third
function, the [M(CO)3]+ (M = 99mTc, Re) moiety, yielded the final trifunctional molecules. The strongly
fluorescent acridine orange, a nuclear targeting agent, has been derivatised with 4-imidazolecarboxylate
as a bidentate ligand and bombesin with an isonitrile group as a monodentate ligand. For cell and
nuclear uptake studies, [Re(L1-BBN)(L2-Ical)(CO)3] type complexes were synthesized and
characterized. For radiopharmaceutical purposes, the 99mTc analogues have been prepared in a stepwise
synthesis. Fluorescence microscopy studies on PC-3 cells, bearing the BBN receptor, showed high and
rapid uptake into the cytoplasm. For the bifunctional molecule, lacking the BBN peptide, no
internalization was observed.
coordination to the [Re(CO)3]+ moiety, switches luminescence
to “on” and enables the visualization of the distribution of the
Introduction
compounds on the subcellular level.7–9 Replacing the rhenium
core with [99mTc(CO)3]+ yielded the homologues targeting radio-
pharmaceutical which can be used for in vivo imaging. Similar
approaches with metal-induced luminescence have been reported
by Parker et al. though without the extension to radionuclides of
e.g. indium.10
The non-invasive visualization of molecular events in vivo and in
vitro is referred to as molecular imaging.1–3 The probes for imaging
include e.g. PET and SPECT radiopharmaceuticals, MRI contrast
agents and photoactive fluorescent compounds. Whereas the latter
two modalities need a stimulus from outside (magnetic field or
spectroscopic excitation) radiopharmaceuticals are active as such.
Radiopharmaceuticals for molecular imaging comprise a targeting
portion which strongly binds to, e.g., receptors. Accumulation
at the target site allows to visualize enhanced expression of cell
receptors or metabolic processes such as glucose based rapid
cell proliferation.4 For cancer diagnosis, targets are typically cell
bound peptide receptors which are often overexpressed in rapidly
proliferating cells. In the near past, there are distinct trends
to combine complementing imaging modalities, in particular
fluorescent and radioactive probes. Such dual, triple or even
quadruple imaging modalities, thus, combine targeting portions,
fluorescent probes for in vitro optical visualization, radionuclides
for in vivo imaging and e.g. magnetic nanoparticles for MRI.5,6
In this context, radiopharmaceuticals may combine a luminescent
and a radioactive probe. These probes may be separated from
each other or be merged into one entity. Valliant et al. used a
special type of isoquinoline based tridentate chelator which, upon
In our own work, we aim at targeting the nucleus of specific
cells with 99mTc or 188Re radiopharmaceuticals. We showed in
vitro that Auger and Koster–Kronig electrons emitted from
99mTc induced double strand breaks in DNA when localized in
its intimate vicinity.11,12 Thus, 99mTc could be used for targeted
therapy and diagnosis in one single compound. Very recently,
Reilly et al. showed that targeted 99mTc Auger electron therapy
with the VEGF-2 K peptide is possible.13 We showed that
the combination of a nucleus targeting agent with a chelator
bound to the [Re(CO)3]+ moiety accumulated in cell nuclei by
confocal fluorescence microscopy.14 Since these conjugates are not
cell specific, we aim at the synthesis of trifunctional imaging
compounds, comprising a receptor specific peptide, a nucleus
targeting, luminescent part and the 99mTc complex. Conceptually,
tri-functionality can be achieved via the metal complex to which
the two modalities are separately coordinated or via a chelator to
which both functions are covalently conjugated. The trifunctional
metal complexes composed of the fluorescent organic dye pyrene,
a nuclear localizing sequence (NLS) peptide and a tridentate
chelator coordinated to the [99mTc(CO)3]+ moiety proved the
concept but the syntheses of those conjugates lacked flexibility
of intercalator and targeting vector variation (Scheme 1).11,15
aInstitute of Inorganic Chemistry, University of Zu¨rich, Winterthurerstr. 190,
8057, Zu¨rich, Switzerland. E-mail: ariel@aci.uzh.ch
bInstitute of Physiology, University of Zu¨rich, Winterthurerstr. 190, 8057,
Zu¨rich, Switzerland
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c0ob00504e
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Scheme 1 Three approaches to trifunctional bioconjugates, (A) cell and nucleus targeting moieties are covalently linked to ligand,11 and (B)
metal-mediated conjugation of both modalities to the [M(CO)3]+ core via the [2 + 1] approach.
Scheme 2 Preparation of an acridine orange based, bifunctional ligand (i) K2CO3, DMF; (ii) 4, toluene, DT; (iii) LiOH, H2O–MeOH.
Since the cell receptor targeting vector should be cleaved in the
intracellular space, conjugates based on the [2 + 1] approach fulfil
the requirement of modular composition.16 In this approach, the
free base of acridine orange (4) to yield 5. Ester hydrolysis of 5
lead then to the free acid 6 (Scheme 2). Compound 6 comprises
the nucleus targeting agent and the bifunctional chelator.
Due to the formation of polyalkylimidazolium side products, the
quarternization yield was low. An alternative route for preparation
of 6 was therefore used. Compound 7 was prepared by the reaction
of AO free base with a large excess of 1,4-dibromobutane. The
alkylation of methyl-4-imidazolecarboxylate 1 with 7 followed by
hydrolysis of the ester group gave a mixture of the regioisomers 6
and 9 (1 : 1; Scheme 3).
These regioisomers could be separated but the mixture could
also used in the next step since the bidentate ligand in 6 coordinates
to [Re(CO)3]+ strongly enough to form a complex stable for
subsequent purification. The reaction of 6 (eventually 6 + 9) with
[Re(H2O)3(CO)3]+ in a H2O–MeOH mixture, afforded the rhenium
complex 10 and the 99mTc homologue 11, respectively (Scheme 4.).
The last step in the formation of the trifunctional complexes
was the introduction of the targeting bombesin vectors 14 or 16.
Bombesin is a 14 amino acid neuropeptide with high affinity for
the gastrin-releasing peptide (GRP) receptor, originally isolated
from frog skin. Even modified bombesin derivatives keep their
high GRP receptor affinity. Bombesin like peptides have been ex-
tensively studied for labeling with radionuclides17,18 since the GRP
receptor is overexpressed in many tumor cell lines. The truncated
peptides 13 and 15 were prepared by standard solid phase synthesis
(SPPS). The reaction of 13 or 15 with isocyanobutyric acid
hydroxysuccinimidyl ester 21 gave the peptide-isonitrile derivatives
14 or 16 in quantitative yields. ESI-MS analysis confirmed the
authenticities of the products. The reaction of 14 or 16 with
complex 10 in acetonitrile/PBS gave the final product 17 or 19
[
99mTc(CO)3]+ core can be labeled with different targeting vectors
and the intercalator moieties, allowing the variability necessary
for drug development. The trifunctional conjugates presented by
Agorastos et al. did not enter the cell nucleus, likely because the
peptide was too strongly bound.14 Thus, we hypothesized that
a peptide bound via a monodentate ligand would facilitate its
release into the intracellular space, leaving the nucleus targeting
part behind. This approach is referred to as the inverted [2 + 1]
approach. We present in this paper a synthetic study for the metal-
mediated conjugation of an acridine orange based intercalator and
a GRP receptor specific peptide according to the inverted [2 + 1]
approach.
Results and discussion
Synthesis. According to the [2 + 1] approach, the nucleus
targeting function should be bound to the [99mTc(CO)3]+ core via a
bidentate chelator. Ideally, this chelator is mono-anionic in order
to give a neutral complex [Re(H2O)(L2-Ical)(CO)3]. The water
ligand is labile and can then be substituted by the monodentate
ligand attached to the cell receptor targeting part. We found that
isocyanides are excellent monodentate ligands. Due to its strongly
fluorescent properties, we have selected acridine orange (AO) as the
intercalator and methyl-4-imidazolecarboxylate 1 as the bidentate
ligand. Compound 6 was prepared in three steps (including ester
hydrolysis). In a first step, the alkylated imidazole 2 (and 3) was
prepared, followed by alkylation of the aromatic nitrogen in the
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Scheme 3 (i) Toluene, DT; (ii) 1, K2CO3, DMF; (iii) LiOH, H2O–MeOH.
Scheme 4 Preparation of mixed ligand [2 + 1] complexes (i) MeOH–H2O; (ii) BnNC, PBS/saline.
(Scheme 5). HPLC-MS analyses showed single peaks with the
correct masses of 804.9 [M+H]2+, 1608.7 [M]+ and 868.9 [M+H]2+,
1736.7 [M]+, respectively.
L2. Second, the monodentate ligand L1 replaced the water ligand.
In this way, coordination of 6 (20 mM) with [99mTc(H2O)3(CO)3]+
at 90 ◦C provided 11 in quantitative yield after 50 min. No
coordination of a second ligand L2 was observed. Subsequent
addition of the ligand BnNC (40 mM) gave the complex 12 after
70 min at r.t. Compound 12 was slightly more lipophilic and,
thus, a longer retention time, 17.8 min, was observed (Fig. 1,
left). Analogous reaction of [99mTc(H2O)(6)(CO)3]+ (11, dashed
Compounds 17 and 19 combine the nuclear-targeting molecules
(AO) and the receptor-targeting portion (BBN), both indepen-
dently bound to the Re or 99mTc (vide infra) center. The chemical
stabilities of these conjugates were assessed by collecting samples
of the compounds incubated in the cell cultivating medium
after different periods of time. HPLC-MS analysis showed the
conjugates to be stable over at least 24 h. No ligand exchange or
decomposition was found and only the oxidation of methionine
to the corresponding sulfoxide was observed.19
◦
line right, retention time 25.0 min) with the peptide 14 at 80 C
gave [99mTc(14)(6)(CO)3]+ (18) in good yield after 60 min. Complex
18 was slightly more hydrophilic (Fig. 1, solid line right, retention
time24.5min). Theauthenticityof 18wasconfirmedbycomparing
the retention time of 18 with that of the Re analogue 17.
Comparable results were obtained for the reaction of the bombesin
analogue 16 (BBN7-14) with 11, yielding 20 with comparable
retention time.
99mTc-labelling studies. The precursor [99mTc(H2O)3(CO)3]+ was
directly prepared from [99mTcO4]- as described.20,21 The concentra-
tion of 99mTc was in the range of 1 nM. The solution was then
buffered with 0.1 M phosphate buffer to pH 7.4. HPLC analyses
with g-detection were performed to quantify the conversion of
the 99mTc. The [2 + 1] mixed ligand synthesis of complexes of
the general type [99mTc (L1)(L2) (CO)3] consists of two consecutive
steps. First, [99mTc(H2O)3(CO)3]+ reacted with the bidentate ligand
The availability of the trifunctional model compounds 17 and
19 and their radiolabeled analogues 18 and 20 serve as examples
for the building block principle of combining individual targeting
agents via binding to a metal core such as the [M(CO)3]+ moiety. In
20 for example, the nucleus targeting agent 6 is combined with the
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Scheme 5 Preparation of the final, bombesin-based trifunctional compounds (i) DMF, Et3N; (ii) MeCN/PBS or PBS/saline.
Fig. 1 Radioactive HPLC traces of [99mTc(H2O)(6)(CO)3]+ 11 (dashed lines) and [99mTc(H2O)3(CO)3]+ (dotted lines). Left—formation of model complex
99mTc(CN-Bn)(6)(CO)3]+ 12 (solid line, Gradient C); Righ—formation of the trifunctional complex [99mTc(15)(6)(CO)3]+ 18 (solid line, Gradient D).
[
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Fig. 2 Uptake of 19 by PC-3 cells, evaluated by fluorescence microscopy. Cells were incubated with 20 mM of 19 for 1 h followed by fixation of cells and
nuclear staining by DAPI. (A) PC-3 cells were loaded with 19 in the cytoplasm (green). (B) Single channel images of the cell for DAPI staining (blue). (C)
Merged image (overlay).
Fig. 3 Uptake of 19 by PC-3. Cells were incubated with complex 19 for 6 h according to the standard protocol. (A) PC-3 cells were loaded with 19 in
the cytoplasm (green), punctuation like structures around the nucleus were observed. (B) DAPI staining (blue). (C) Merged image (overlay).
receptor specific peptide 15 both representing the two biological
functions. The third function, the99mTc core, serves in this system as
the imaging or therapeutic portion. Each of these functions can be
altered individually which allows for optimization of a particular
agent without fully new syntheses. Admittedly, a two step synthesis
to a trifunctional radiopharmaceutical is not ideal for routine
application; however, it suffices the drug-finding concept in the
first moment.22
a similar time dependent behavior with 19 (see ESI†). It seems that
the two compounds, regardless of their slightly different peptides,
behave in a comparable way in the cells.
In contrast, in the absence of the receptor targeting vector (pep-
tides BBN) such as in model complex 10, almost no uptake was
observed, even after extended incubation times (Fig. 4). Similar
results were obtained for the pure acridine orange derivative 6
which carries no metal complex. This selective behavior of the full
trifunctional cellular probes clearly underscores the requirement
for a targeting vector in the trifunctional system and is in support
of the cell specificity concept. The absence of uptake with 10 (or
6) alone showed cell fluorescence to be due to internalization of
the complete trifunctional conjugates and not just by eventual
complex decomposition prior to the uptake.
To underline the cell specificity concept, similar studies were
performed on a B16-BL6 mouse melanoma cell line which doesn’t
express the GRP receptor. While there was virtually no uptake
of compound 19 in B16-BL6 cells, PC-3 cells were loaded with
19 in the cytoplasm, displaying the punctuate accumulation at
specific sites as described before (Fig. 5). The uptake of 19 by the
cells was significantly suppressed in B16-BL6 cells, indicating that
this complex could not cross the cell membrane. The uptake of
complex 19 by PC-3 cells was consistent with the presence of the
GRP receptor, indicating a receptor-mediated uptake.
Cell uptake studies. To study the cell uptake of the tri-
functional compounds by confocal fluorescence microscopy, we
selected the human prostate adenocarcinoma cell line PC-3 which
does express the GRP receptor23 and B16-BL6 mouse melanoma
cell line with no expression of the GRP receptor. The PC-3 cell
line was used for targeting studies with the model complex 10 and
the BBN derivatives 17 and 19. The uptake of complex 19 was
studied as described in the experimental section. To localize the
compounds in the cells and eventually in the nucleus, we stained
the nuclei with DAPI (4¢,6-diamidino-2-phenylindol). The PC-3
cells were exposed to 19 (20 mM) and, after 1 h, the distribution
of 19 in the cells was detected by fluorescence microscopy (Fig. 2).
Complex 19 accumulated in the cell and most of the fluorescence
was localized in the cytoplasm.
Incubating the cells with 19 for 6 h revealed an interesting
feature. The accumulation of 19 in the cells’ cytoplasm produced
a punctate like accumulation at distinct sites in the cell around the
nuclei (Fig. 3). A similar heterogeneous distribution of peptides,
derivatised with fluorescent ruthenium complexes, has recently
been described.24 In some cases, accumulation only took place in
one particular region of the cell. Similar studies with 17 exhibited
Conclusion
An efficient development of cell specific, nucleus targeting agents
for molecular imaging and therapy requires a building block
concept. In this work, we have applied the [2 + 1] concept for the
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Fig. 4 Uptake of 10 by PC-3. Cells were incubated for 12 h according to the standard protocol. (A) Complex stain (green). (B) DAPI staining (blue).
(C) Merged image (overlay).
uptake), they underline the feasibility of the concept and reveal
also the deficiencies of specific compounds. In particular, while
the stable bifunctional complex did not penetrate the cell, the full
trifunctional complexes were taken up into cells with the GRP
receptor. The strength of the concept is the convenient alteration
of any of its functions. Currently, detailed studies with 99mTc
complexes bearing functionalities with improved nuclear uptake
are being studied.
Experimental section
All solvents were of puriss or p.a. quality. The commercially avail-
able reagents were used as received without further purification.
Isocyanobutyric acid hydroxysuccinimidyl ester (21) was a gift
from Dr Hans-Ju¨rgen Pietzsch, Forschungszentrum Rossendorf,
Germany and is gratefully acknowledged. TLC on aluminium-
based silica gel plates (silica gel 60 F254) from Merck, Flash
chromatography with Flashmaster Solo (Argonaut) with Merck
silica gel 60 (0.040–0.063 mm). Samples were applied as almost
saturated solutions in the appropriate solvent. 1H NMR and 13
C
NMR spectra were recorded on Varian Gemini 200 and Bruker
400 spectrometers at 200 and 50 MHz, and 400 and 100 MHz
respectively. The reported chemical shifts (in d) are relative to
the solvent protons and carbons. HPLC-MS (ESI) spectra were
measured on a Bruker HCT spectrometer (Bruker) with Aquinity
UPLC (Waters), using a Macherey-Nagel EC 250/3 Nucleodur
C18 Gravity 5 mm. HPLC solvents were 0.1% formic acid (solvent
A) and methanol (solvent B). The gradients used for analyses
were: Gradient A: 0–5 min 90% A, 5–17 min 90–0% A, 17–25
min 0% A, 25–27 min 0–90% A, 27–35 min 90% A, flow 0.3 mL
min-1; Gradient B: 0–2 min 90% A, 2–12 min 90–0% A, 12–17
min 0% A, 17–18 min 0–90% A, 18–25 min 90% A, flow 0.25 mL
min-1. HPLC analyses were performed on a Merck L7000 system
using a Macherey-Nagel EC 250/3 Nucleodur C18 Gravity 5 mm
for radioactive compounds. HPLC solvents: 0.1% trifluoroacetic
acid (solvent A) and MeOH or MeCN HPLC grade (solvent B).
Gradient C (MeOH): 0–5 min 90% A, 5–15 min 90–0% A, 15–20
min 0% A, 20–22 min 0–90% A, 22–30 min 90% A. Gradient D
(MeCN): 0–5 min 90% A, 5–20 min 90–60% A, 20–23 min 60% A,
23–28 min 0% A, 28–33 min 0% A, 33–34 min 0–90% A, 34–40 min
90% A. Flow rates: 0.5 mL min-1, detection with a g-detector for
the radiolabeled compounds. Preparative HPLC was performed
on a Varian Pro Star system by using either a Macherey-Nagel
VP 250/21 Nucleodur C18 Gravity 5 mm or a Macherey-Nagel
Fig. 5 Uptake of 19 by PC-3 and B16-BL6. Cells were incubated with
complex 19 (20 mM) for 6 h, then the medium was changed and the cells
were kept in the fresh medium for 24 h, fixed and stained according to the
standard protocol. (A) PC-3 cells were loaded with 19 in the cytoplasm
(green), punctuation around the nucleus (DAPI stain, blue) observed. (B)
B16-F1 cells showed no uptake (background level green fluorescence).
formation of trifunctional radiopharmaceuticals. Fluorescence
microscopy with Re compounds allows for localization of the
compounds on the sub-cellular level whereas the analogous 99mTc
compounds can be used for in vivo imaging. Although compounds
19 and 20 studied herein are not yet ideal (too little nucleus
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VP 250/40 Nucleosil 100–7 C18 column with flow rates of
12 mL min-1 and 40 mL min-1, respectively. The solvents were
0.1% trifluoroacetic acid (solvent A) and methanol or acetonitrile
(solvent B). The exact mass measurements were obtained using
peak top or centroid depending on the peak shape for each analyte
(see ESI†). The elementary analyses for some compounds were not
performed due to the various contents of trifluoroacetate after
purification with the preparative HPLC. Thus, to be consistent,
the M.W. and concentrations of the compounds were calculated
without counter-ions (e.g. as drawn in the schemes). If not specified
in the scheme, the counter-ions are trifluoroacetates.
flash chromatography (gradient, CH2Cl2 to CH2Cl2–MeOH–
isopropylamine 100 : 5 : 1) afforded two fractions. The second
fraction contained 7 mg of 5 (7%) as an orange powder. 1H NMR
(CD3OD, 400 MHz): d 8.55 (s, 1H), 7.90–7.74 (m, 4H), 7.53–7.45
(dd, 3J = 9.3 Hz, 4J = 2.0 Hz, 2H), 6.50 (s, 2H), 4.64 (t, 3J = 8.0 Hz,
3
2H), 4.14 (t, J = 6.6 Hz, 2H), 3.75 (s, 3H), 3.20 (s, 12H), 2.12–
2.02 (m, 2H), 1.92–1.80 (m, 2H). HPLC-MS (ESI, Gradient A)
tR: 15.7 min; m/z: 446.3 (100) [M]+. Calcd for C26H32N5O2 : 446.3
+
(100%). The first fraction contained 3,6-bis(dimethylamino)-10-
methylacridinium. Analyses were similar to the literature.25
10-(4-(4-Carboxy-1H -imidazol-1-yl)butyl)-3,6-bis(dimethyl-
amino)acridinium (6). 5 (5 mg, 0.011 mmol) was dissolved in
MeOH (1 mL) and a solution of LiOH in water (0.5 mL, 1 M)
was added to the mixture. The reaction was stirred and heated
to 50 ◦C for 12 h. After the neutralization with HCl (0.5 M), the
solvent was removed in vacuo. The crude product was purified by
preparative HPLC to give 6 as an orange powder in a yield of
Cell culture and complex uptake. Human prostate cancer cells,
PC-3 were incubated in F12 K medium supplemented with 10%
fetal bovine serum. B16-BL6 mouse melanoma cells were cultured
in RPMI-1640 supplemented with 10% fetal bovine serum. For
microscopy, cells were cultured overnight on 4-chamber slides
(Nunc) in which ca. 50 000 cells were plated. The next day, cells
were washed and fresh medium containing complex 19, 17, 10
or 6 (20 mM) was added. Cells were incubated with the complex
for 1–24 h. After loading with a complex, cells were washed with
phosphate buffer saline (PBS) and fixed in 4% paraformaldehyde
for 10 min at r.t. After three washings with PBS, cells were
incubated in 1 mg mL-1 4¢,6-diamidino-2-phenylindole (DAPI)
for nuclear staining for 10 min at r.t. Cells were washed three
times with phosphate-buffered saline (PBS), chambers from the
slides were detached and cells were covered with coverslips in
Prolong Mounting medium (Invitrogen). In order to get well
resolved images for the cell specificity studies (B16-BL6 vs. PC-
3), the loaded cells were kept for 24 h in the fresh medium prior to
fixation. Slides were evaluated on Zeiss Axiovert 200 m with the
1
(4.6 mg, 95%). H NMR (CD3OD, 400 MHz): d 8.67 (bs, 1H),
8.64 (s, 1H), 8.10 (bs, 1H), 7.88 (d, 3J = 9.2 Hz, 2H), 7.24 (d, 3J =
8.9 Hz, 2H), 6.62 (s, 2H), 4.74 (bt, 3J = 8 Hz, 2H), 4.29 (bt, 3J =
6.5 Hz, 2H), 3.27 (s, 12H), 2.26–2.09 (m, 2H), 2.06–1.91 (m, 2H).
HPLC-MS (ESI, Gradient A) tR: 14.5 min; m/z: 432.2 (100) [M]+.
+
Calcd for C25H30N5O2 : 432.2 (100%). The mixture of 6 and 9
was obtained according to the preparation 2/3 using compound 7
instead of 1,4-dibromobutane, followed by hydrolysis as for 6. The
regioisomers were separated using preparative HPLC to afford 6
and 9. For analyses of 9 see the ESI.†
10-(4-Bromobutyl)-3,6-bis(dimethylamino)acridinium bromide
(7). Acridine orange free base 4 (50 mg, 0.19 mmol) was
dissolved in toluene (dry, 5 mL) and 1,4-bromobutane (0.5 ml,
912 mg, 4.22 mmol). The mixture was heated to reflux. After
24 h, the reaction mixture was diluted with toluene (15 mL)
and the precipitate filtered. The crude product was washed with
toluene and toluene with few drops of NH4OH. The residue was
dissolved in CH2Cl2 and the orange solution was diluted with
hexane until product precipitated. The solution was basified with
a few drops of NH4OH, stirred for 10 min at r.t. and filtered.
Finally, the precipitate was washed with hexane and dried under
corresponding fluorescence filters for DAPI (lex = 360 nm, lem
420 nm) and acridine orange (lex = 496 nm, lem = 525 nm).
=
Methyl-1-(4-bromobutyl)-1H-imidazole-4-carboxylate (2).
A
suspension of methyl-4-imidazolecarboxylate (1) (200 mg,
1.58 mmol) and K2CO3 (700 mg, 5 mmol, dry) in DMF (10 mL,
dry) was stirred at r.t. under N2. After 10 min, 1,4-dibromobutane
(1.9 ml, 3.4 g, 15.8 mmol) was added and the mixture stirred and
heated to 50 ◦C under N2. After 16 h, the solvent was removed
under HV. The residue was dissolved in 15 mL of CH2Cl2, filtered
over Celite, and dried again. Purification by flash chromatography
(gradient, hexane to EtOAc) afforded two main fractions. The
second fraction contained 130 mg of 1,4 regioisomer 2 (32%) as a
light yellow oil. The compound was immediately used in the next
step. 1H NMR (CDCl3, 400 MHz) d 7.62 (s, 1H), 7.49 (s, 1H), 4.02
(t,3J = 7.0 Hz, 2H), 3.88 (s, 3H), 3.40 (t,3J = 6.4 Hz, 2H), 2.02–1.95
(m, 2H), 1.90–1.80 (m, 2H). MS (ESI) m/z (%) 261.4 (100), 263.4
1
HV to give 7 (70 mg, 77%) as a dark orange powder. H NMR
(CD3OD + CDCl3, 200 MHz): d 8.61 (s, 1H), 7.88 (d, 3J = 9.2 Hz,
3
2H), 7.14 (d,3J = 9.2 Hz, 2H), 6.77 (bs, 2H), 4.81 (bt, J = 8 Hz,
2H), 3.72 (t,3J = 5.8 Hz, 2H), 3.35 (s, 12H), 2.45–2.00 (m, 4H).
HPLC-MS (ESI, Gradient A) tR: 16.6 min; ESI-MS: m/z: 402.1
(100), 400.1 (98) [M-Br]+. Calcd for C21H27BrN3 : 402.1 (100.0%),
+
400.1 (99.9%). Elemental analysis (%) calcd for C21H27Br2N3: C
52.41, H 5.65, N 8.73; found: C 53.05, H 5.55, N 8.47.
(98) [M + H]+. Calcd for C9H14BrN2O2 : 261.0 (100.0%), 263.0
+
[Re(H2O)(6)(CO)3]+ (10). To
a solution of 6 (4 mg,
(98.2%). Elemental analysis (%) calcd for C9H13BrN2O2: C 41.40,
H 5.02, N 10.73; found: C 41.75, H 4.81, N 10.94. TLC Rf 0.13
(EtOAc). The first fraction contained 113 mg of 1,5 regioisomer 3
(27%) as a light yellow oil. For analyses see the ESI.†
0.0092 mmol) in CH3OH–H2O (1 mL, 1 : 1) [NEt4]2[ReBr3(CO)3]
(8 mg, 0.01 mmol) was added. The mixture was stirred at r.t.
under N2 for 4 h. A precipitate was filtered, washed with water and
hexane, and dried in desiccator. The crude product was purified
by preparative HPLC to give 10 as orange powder in a yield of 5
mg (61%). 1H NMR (CD3CN, 400 MHz): d 8.55 (s, 1H), 7.89 (s,
1H), 7.83 (d, 3J = 9.4 Hz, 2H), 7.47 (s, 1H), 7.16 (dd, 3J = 9.3 Hz,
4J = 2.1 Hz, 2H), 6.51 (s, 2H), 4.59 (bt, 3J = 8 Hz, 2H), 4.12 (t, 3J =
6.8 Hz, 2H), 3.22 (s, 12H), 2.09 (m, 2H), 1.90 (m, 2H). HPLC-MS
(ESI, Gradient B) tR: 12.2 min; m/z: 702.3 (100) [M-H2O]+, 700.3
3,6-Bis(dimethylamino)-10-(4-(4-(methoxycarbonyl)-1H-imida-
zol-1-yl)butyl)acridinium (5).
A solution of 2 (120 mg,
0.46 mmol) and acridine orange free base 4 (60 mg, 0.22 mmol)
in toluene (10 mL) was stirred under N2, for 24 h at 120 ◦C.
The formed precipitate was filtered and washed with hex-
ane and dried in vacuo. Purification of the crude product by
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(57) [M-H2O]+. Calcd for C28H29N5O5Re+: 702.2 (100.0%), 700.2
(57.6%).
(ESI, Gradient B) tR: 12.2 min; m/z: 868.9 (100) [M+H]2+, 579.6
(21) [M+2H]3+. Calcd for C76H99N19O15ReS+: 1736.7 (100.0%),
1737.7 (79.8%).
Bombesins WAVGHLM (13) and QWAVGHLM (15). Trun-
cated bombesins 13 (sequence: WAVGHLM) or 15 (sequence:
QWAVGHLM) were synthesized SPPS according to a procedure
previously reported.14 The products were then purified by reverse
phase HPLC on a preparative C18 column (Macherey-Nagel VP
250/40 Nucleosil 100–7 C18 column) using a gradient of 5–55%
acetonitrile in water (+ 0.1% TFA) over 45 min and a flow of
40 mL min-1. Fractions with the peptides were lyo◦philized; pure
peptides were kept under N2 in the freezer at -25 C. 13 and 15
were obtained as white solids. 13: HPLC-MS (ESI, Gradient A)
tR: 13.3 min; positive mode m/z: 812.5 (100) [M+H]+, 406.7 (56)
[M+2H]2+; negative mode m/z: 810.5 (56) [M - H]-, 924.5 (100)
[M+TFA]-. Calcd for C38H58N11O7S+: 812.4 (100.0%). 15: HPLC-
MS (ESI, Gradient B) tR: 10.6 min; positive mode m/z: 940.7 (100)
[M+H]+, 470.9 (98) [M+2H]2+; negative mode m/z: 938.7 (100) [M
- H]-. Calcd for C43H66N13O9S+: 940.5 (100.0%).
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1078 | Org. Biomol. Chem., 2011, 9, 1071–1078
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The Royal Society of Chemistry 2011
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