One to chelate them all: Investigation of a versatile, bifunctional chelator for 64Cu,99mTc, Re and Co

Article abstract of DOI:10.1039/c0dt01458c

We describe the synthesis of the dip (di-picolyl-carboxylate) bifunctional chelator system, capable of fast coordination of Cu²⁺, ⁶⁴Cu²⁺ and Co²⁺, as well as the [M(CO) ₃]⁺-core (M = ⁹

Full text of DOI:10.1039/c0dt01458c

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Radiometallated peptides for molecular imaging and targeted therapy
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Towards translation of 212Pb as a clinical therapeutic; getting the lead in!
Kwon Yong and Martin W. Brechbiel
Dalton Trans., 2011, DOI: 10.1039/C0DT01387K
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First dinuclear Re/Tc complex as a potential bimodal Optical/SPECT molecular imaging agent
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Synthesis, cytotoxicity and cellular uptake studies of N3 functionalized Re(CO)3 thymidine
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Dalton Trans., 2011, DOI: 10.1039/C0DT01452D
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PAPER
One to chelate them all: investigation of a versatile, bifunctional chelator for
64Cu, 99mTc, Re and Co†
Eszter Boros,a,b Yi-Heng Scott Lin,a Cara L. Ferreira,c Brian O. Patrick,a Urs O. Ha¨feli,d Michael J. Adam*b
and Chris Orvig*a
Received 23rd October 2010, Accepted 23rd December 2010
DOI: 10.1039/c0dt01458c
We describe the synthesis of the dip (di-picolyl-carboxylate) bifunctional chelator system, capable of
fast coordination of Cu2+, 64Cu2+ and Co2+, as well as the [M(CO)3]+-core (M = 99mTc, Re); it displays a
variety of binding modes—tridentate when protected, tetradentate when deprotected. Syntheses of both
the benzyl-nitro derivative and the benzyl-amino derivatives are described. The latter was coupled to
biotin to show the viability of the system for functionalization with biomolecules. Besides coordination
chemistry with stable isotopes, we also present labelling data with 64Cu and 99mTc, as well as in vitro
stability studies.
environment preferences. All metals investigated in this study have
Introduction
radioisotopes that are of considerable interest for either imaging
+
or therapy. 64Cu (t1/2 = 12.7 h, b 17.4% Emax = 0.656 MeV, b- 39%,
The development of new radiopharmaceuticals for imaging and
therapy of disease is a constantly burgeoning field.1,2 Due to the
Emax = 0.573 MeV) is of high current interest for both positron
availability and properties of the 99mTc generator for the past three
decades, the development of novel imaging agents has centred
around this particular isotope3 and has been strongly affected
by the recent shortage of its parent 99Mo. We, like many other
research groups, have recognized a need for closer investigation
of potential imaging agents based on alternative isotopes.1,4 Of
specific importance is the ability to pick and choose isotopes
according to their properties such as t1/2, mode and energy of
decay, range of emitted particle, or even availability.5,6 The need for
different chelators specific to each radiometal, or particular leaving
groups for the incorporation of radiohalogens or 11C, imposes a
non-negligible additional difficulty and often obviates the fast and
simple synthesis of analogous targeting vectors labelled with a
different radionuclide.
emission tomography (PET) and radiotherapy.7 The longer half-
life is more applicable to developing PET agents with larger
biomolecules, such as monoclonal antibodies, that may require
longer circulation times before imaging to achieve optimal target
+
uptake.8 55Co (t1/2 = 17.6 h, b 100% Emax = 0.54 MeV) is a
cyclotron produced isotope suitable for PET; however, only a few
55Co labelled compounds have been investigated for their potential
as imaging agents.9,10 99mTc is the most commonly used isotope in
nuclear medicine, partly due to its near ideal physical properties
(t1/2 = 6 h, g = 140 keV).3 The[99mTc(CO)3]+ core canbe conveniently
prepared by the Isolink kit developed by Alberto and co-workers11
and serves as a scaffold which can be easily chelated through
exchange of the three labile H2O molecules to form highly stable
complexes with tridentate ligands.12 The stable isotopes of Re serve
as cold congeners for the radioactive 99mTc and are used to optimize
cold chemistry and perform standard characterization techniques,
while 186/188Re is attractive for therapeutic b- applications. 186Re
(t1/2 = 90 h, Eb = 1.07 MeV, max. range in tissue = 5 mm,
Eg = 137 keV) is suitable for treating smaller tumours, whereas
188Re (t1/2 = 17 h, Eb = 2.1 MeV, range max. range in tissue =
11 mm, Eg = 155 keV) is suitable for treating larger tumours.
186Re is cyclotron produced, while 188Re can be furnished with high
specific activity from a 188W/188Re generator system in the form of
Herein, we describe our efforts in design and synthesis of
versatile, universal chelate systems, competent to label a variety of
radiometals of different oxidation states and varying coordination
aMedicinal Inorganic Chemistry Group, Department of Chemistry, The
University of British Columbia, 2036 Main Mall, Vancouver, BC, V6T 1Z1,
Canada. E-mail: orvig@chem.ubc.ca; Fax: +01 604 822 2847; Tel: +01 604
822 4449
bTRIUMF, 4004 Wesbrook Mall, Vancouver, BC, V6T 2A3, Canada.
E-mail: adam@triumf.ca
[
188ReO4]-.13,14 All (radio-)metals described have a preference for
cNORDION, 4004 Wesbrook Mall, Vancouver, BC, V6T 2A3, Canada
dFaculty of Pharmaceutical Sciences, The University of British Columbia,
2146 East Mall, Vancouver, BC, V6T 1Z3, Canada
being coordinated by polyamino-carboxylate ligands of a variety
of denticities.
† Electronic supplementary information (ESI) available: Detailed infor-
mation on solid state structures, key NMR spectra, radio-HPLC and
radio-TLC traces. CCDC reference numbers 798347 and 798348. For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c0dt01458c
Despite the fact that most acyclic ligands have low stability
with Cu(II),15 select examples such as the thiosemicarbazone
H2ATSM (diacetyl-bis(N-4-methylthiosemicarbazone)) still are a
viable option for the stable chelation of this metal.16 We were
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©

Fig. 1 Ligand systems dpa, Me2dipin and H2dipin. Coordination sites are indicated with arrows for each ligand system.
inspired by the very versatile and elegant SAAC (single amino acid
chelate) approach of Valliant, Babich, Zubieta and coworkers,17
our own recent work on novel 68Ga chelators18 and some select
examples of multifunctional chelation systems.19,20
benzylamine derivative would be converted to an aminobenzyl-
amine fragment, and the phenylamine could then be coupled to
a biomolecule. Only the benzylamine should react during the
reductive amination reaction, so it is important to protect the
phenylamine during this step. Two strategies have been followed
to accomplish this.
We decided to investigate the “dip” (di-picolyl-carboxylate)
ligand system which contains two pyridine carboxylate moieties
joined by a benzyl amine functionality for derivatization. The
chelate system itself was previously investigated for Pb2+ and found
to bind this particular metal ion in a pentagonal, planar fashion.21
We investigated different chelation modes of dip, namely square
planar for the divalent metals Cu2+ and Co2+, as well as fac-
tridentate for the Re(CO)3 and Tc(CO)3-cores with a model chelate
(3, 4) (Fig. 1). We then also explored the viability of the system
for simple functionalization through synthesis and coordination
of the corresponding biotin derivatives (7, 8). Biotin is a water
soluble molecule also known as vitamin H. In vivo, biotin acts
as a coenzyme involved in metabolism and in the production of
important biomolecules such as fatty acids and antibodies. At the
same time, its very high affinity towards avidin and streptavidin
(Kd ~10-15M) has led to many biochemical applications that are
dependent on this strong affinity.22 The biotin-avidin interaction
has also led to investigations in the area of targeted cancer
radiotherapy.23 Conjugation of our bifunctional chelate to biotin
is therefore highly relevant, and serves as a convenient proof of
principle for the feasibility of our design.
Our first synthesis strategy involved the use of
a
4-nitrobenzylamine fragment to form 3 (Me2dipin, Scheme 1).
Sodium triacetoxyborohydride is a mild reducing agent that was
strong enough to reduce the imine intermediate, but not too
strong to reduce the methyl ester. Since 4-nitrobenzylamine was
purchased as a hydrochloride salt, basifying the salt with sodium
hydroxide was initially performed to form the corresponding
amine. The reductive amination reaction was run overnight and
quenched with saturated aqueous Na2CO3. Since the product was
minimally soluble in methanol, it was found that recrystallization
of the crude product mixture was possible with 4 : 1 MeOH–
DCM, affording the pure product 3 as brown needle-like crystals.
Subsequent deprotection of the methylester protection group
with LiOH afforded the desired model compound 4 (H2dipin)
quantitatively.
To afford the easily derivatized benzylamine, however, reducing
the nitro group of 3 to form 6 proved to be unsuccessful. We
R
initially attempted the reduction using hydrazine with RANEYꢀ
nickel as the catalyst. ESI-MS evidenced formation of the product,
1
but the H NMR spectrum showed broadened peaks, suggesting
possible coordination of the chelate to Ni(II). The same reduction
reaction was attempted again using H2 with Pd/C catalyst. Low to
moderate pressure of H2 did not drive the reaction to a significant
yield, whereas higher pressure (70 psi) led to cleavage of the
nitrobenzylamine fragment, as suggested by both H-NMR and
ESI-MS. Hence, the reduction method for this specific nitro group
was concluded to be ineffective.
The second, more successful strategy involved Fmoc-protected
4-aminobenzylamine as the starting material (Scheme 1). 4-
Aminobenzylamine can be selectively protected by Fmoc at
the phenylamine position to produce 4-(Aminomethyl)-N-(9-
fluorenylmethoxycarbonyl)phenylamine as a hydrochloride salt.26
Basic extraction was then performed to obtain the corresponding
free amine. This was reacted with 2 equivalents of 2 to form 5 via
a double reductive amination reaction. The product was purified
by column chromatography, yield 57%. Finally, deprotection with
20% piperidine in DMF afforded the intended 6 in 92%.
Results and discussion
Synthesis and characterization
1
A double reductive amination reaction was chosen to join the two
functional ends of the proposed bifunctional chelate. Commer-
cially available dipicolinic acid, which can be turned into a partially
oxidized aldehyde with a methyl ester protected carboxylate
in three previously reported reaction steps, would be ideal for
this purpose.24 NaBH4 was used as a reducing agent to afford
hydroxymethyl pyridine-2-carboxylic acid methyl ester (1) through
partial reduction. The yield of this step was 55%. The following
step involved oxidation of the alcohol group to its corresponding
aldehyde 2, and this reaction used selenium dioxide in 1,4-dioxane
as the oxidizing condition. After the reaction, the selenium salt
and impurities were removed by column chromatography (silica,
CHCl3), yielding the clean product (48%).25
To synthesize the biotin-conjugated fragment, the carboxylic
acid end of biotin was first activated by the use of tetraflu-
orophenyl ester following literature conditions.22 The strong
Compound 2 can then react with a benzylamine derivative,
undergoing a double reductive amination reaction, to form the
framework of the intended bifunctional chelate.17 Ultimately, this
6254 | Dalton Trans., 2011, 40, 6253–6259
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Scheme 1 (a) 4-Nitrobenzylamine, NaBH(OAc)3, DCE, reflux, 22 h. (b) 4-(Aminomethyl)-N-(9-fluorenyl methoxycarbonyl)phenylamine, NaBH(OAc)3,
DCE, reflux, 12 h. (c) 20% piperidine, DMF, 30 min. (d) LiOH, 3 : 1 THF–H2O.
Scheme 2 Synthesis of biotinylated conjugates 7 and 8. (e) Biotin-TFP, TEA, DMF, 60 ◦C, 16 h, (f) LiOH, 3 : 1 THF–H2O.
electron-withdrawing ability of the tetrafluorophenyl group weak-
ens the carboxylate C–O bond, making the carbonyl particularly
susceptible to nucleophilic attacks.
of the solution intensified during neutralization of pH. Copper(II)
acetate was used as the starting material of copper complex
formation. Copper(II) acetate is a dark green crystal, and when
this precursor was added to the chelator solution, the product
complex precipitated, forming a cloudy suspension. Evaporation
of the solvent gave the copper complex which was confirmed by
high resolution mass spectrometry and for [Cu(4)(H2O)] by X-ray
crystal structure determination (Fig. 2). For the synthesis of the
cobalt complexes, cobalt(II) chloride was used. This complex is
pink in hydrated form, but blue in the anhydrous form. Similarly,
the product complex was light purple in hydrated form, but
bluish-green after removal of water in vacuo. The IR spectrum
of the cobalt complexes showed a broad peak around 3300 cm-1,
suggesting that water ligands were coordinated to the cobalt centre.
Successful complexation was confirmed by high-resolution mass
spectrometry. Because both Cu(II) and Co(II) are paramagnetic,
NMR was not used to characterize the product complexes.
Compounds 3 and 7 were used for the coordination of
the Re(CO)3-core.27,28 While the carboxylates remain protected
and cannot act as binding groups, the two pyridyl nitrogen
atoms together with the aliphatic nitrogen form the widely
explored and popular dpa (dipicolylamine) ligand system.
To afford [Re(3)(CO)3]Br, reactants were stirred in a 1 : 1
methanol–dichloromethane mixture with equimolar amounts of
[Re(CO)3(H2O)3]Br and ligand over night at 40 ◦C. Unreacted
ligand was removed from the crude through trituration with
Once biotin-TFP was prepared, it was then reacted with 6 in
DMF (Scheme 2). Triethylamine was also added to maintain the
basic condition that would be ideal for the coupling reaction.22
After overnight stirring at 70 ◦C, the product was identified by
ESI-MS; the crude solid was triturated with THF to remove
impurities and to afford the pure product 7 (Me2dipiam-biotin)
as a yellow solid. Compound 8 (H2dipiam-biotin) was prepared
through LiOH deprotection of the methyl esters.
Cold coordination chemistry
To synthesize the copper, cobalt, and rhenium complexes, the
chelator was mixed with the corresponding precursor metal
complex to form the desired product complex via a ligand
substitution reaction. Compounds 4 and 8 were prepared by
hydrolyzing 3/7 with 4 equivalents of LiOH (Scheme 1 and
Scheme 2). This frees the picolinate groups for coordination.
To synthesize copper and cobalt complexes, 4/8 was acidified
in a MeOH–H2O mixture. The initial acidic condition prevents
the hydrolysis of copper and cobalt to form insoluble copper
or cobalt hydroxides. The solution was then neutralized to pH
7–8 after addition of complex precursors. Upon addition of the
chelator, an immediate color change was observed, and the color
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©

Fig. 2 ORTEP representations of [Cu(4)(H2O)] (left) and [Re(CO)3(3)]+ (right); the counter ion [Re(CO)3Br3]2- (on the right) and hydrogen atoms are
omitted for clarity.
dichloromethane to give the corresponding clean tridentate rhe-
nium complex in good yield. In the case of [Re(7)(CO)3]Br,
reactants were stirred in methanol overnight at 60 ◦C. The crude
reaction mixture was purified using preparative HPLC. Metal ion
coordination of the tridentate ligands could be verified by shifts in
peaks in both the 1H and 13C NMR spectra, as well as confirmation
of the predicted mass of the cation [M+] with high resolution mass
spectrometry.
water mixture due to poor solubility of the ligand in protic solvents,
yielding 72% product after reaction, with Rf 0.8. Due to increased
solubility of 7 in protic solvents, labelling for this ligand was
performed in a 3 : 7 methanol–water mixture, yielding 85% product
with an Rf of 0.57. Both complexes were stirred in 0.1 M solution
of histidine and cysteine at 37 ◦C for 24 h, and were found to be
fully stable against decomposition (Table 1).
64Cu labelling complex stability
X-Ray crystallography
A solution of 64Cu was added to a 10-4 M solution of ligand (4 or
8, both in aqueous solutions) at pH 5. The reaction was analyzed
by HPLC after 10 min at room temperature. 64Cu(4) was found to
be formed with a yield of 95% with Rt = 10.94 min. 64Cu(8) was
found to be formed with a yield of 91% with Rt = 8.1 min. To
evaluate complex stability, an aliquot of the reaction was added◦to
a solution of mouse serum. The mixture was incubated at 37 C
and analyzed by HPLC after 1 h. For 64Cu(4) 78% serum binding
was found after 1 h, while in the case of 64Cu(8), 52% activity
was found to be bound to serum after the same incubation time
(Table 1).
Cu(4) pale green solids were dissolved in a DCM–MeOH mixture,
and crystals were obtained by slow evaporation (Fig. 2, left). The
relevant bond angles (see ESI)† show that the complex is of square
pyramidal geometry, and the four donor atoms from the chelator
are sitting on a distorted square plane. The bond length between
Cu and any of the 4 donor atoms of the chelator is roughly the
same; however, the Cu–N bond lengths are slightly longer than
the Cu–O bond lengths, suggesting that Cu–O bond strength
may be slightly stronger than that of Cu–N. The bond length
between Cu and the apical water oxygen atom is longer than the
other coordinate bonds, suggesting that the water molecule may
be loosely coordinated to the metal centre. The axial elongation is
typical of the Jahn–Teller distortion observed for Cu(II) complexes
andis alsoinagreementwithpreviouslyreportedsquarepyramidal
Cu(II) complexes. The axially coordinated water molecule could
be another indicator for the instability of this particular ligand
system in vitro, since it could be easily displaced by serum proteins
facilitating the transchelation of the metal out of the dipin ligand.
For the structure of [Re(CO)3(3)]+ (Fig. 2, right), the colour-
less crude solid was dissolved in MeOH and crystals were
obtained by slow evaporation. The complex co-crystallized
with [Re(CO)3Br3]2- as the counter ion for two molecules of
[Re(CO)3(3)]+. The observed bond angles and lengths (see ESI)†
point to a distorted octahedral structure with all Re–N bond
99mTc labelling and complex stability
The organometallic precursor [99mTc(H2O)3(CO)3]+ was synthe-
sized via heating, using the Isolink kit. When the mixture was
cool, the pH was adjusted to approximately 7. A 10-4 M solution
of ligand was mixed with [99mTc(H2O)3(CO)3]+ in a sealed vial and
heated to reflux for 40 min. Reaction monitoring was performed by
radio-TLC. Labelling with 3 was performed in a 1 : 1 acetonitrile–
Table 1 Data from labelling studies of all ligands
Ligand
Isotope
Labelling yield
Stability (time)
99mTc
99mTc
64Cu
64Cu
72%
85%
95%
91%
99% (24 h)
99% (24 h)
22% (1 h)
48% (1 h)
˚
lengths close to 2.2 A, which is characteristic for bipyridyl com-
3
7
4
8
plexes of the Re(CO)3-core, indicating that the stable coordination
of this scaffold is not influenced by the two adjacent methyl ester
functionalities.
6256 | Dalton Trans., 2011, 40, 6253–6259
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©

removed from the filtrate by rotary evaporation. A mixture of
white and red solids remained. The crude product was purified by
column chromatography (SiO2, chloroform) as an off-white solid
Conclusions
We have successfully synthesized the model ligands 3 and 4 as well
as the corresponding bifunctional versions 7 and 8. Corresponding
cold complexes with copper(II), cobalt(II) and tricarbonylrhe-
nium(I) were prepared in satisfying yields. Radiolabelling with 64Cu
was fast and efficient with both 4 and 8. 99mTc(CO)3 complexes with
both 3 and 7 were synthesized under standard labelling conditions.
While the stability of the 99mTc(CO)3 complexes was satisfactory,
the 64Cu complexes were found to be not stable enough for further
development or in vivo applications. The acyclic, tetradentate
coordination mode of the dip ligand system was shown to be a non
ideal chelation scaffold, despite the use of strong donor atoms as
ligands. We are currently investigating other multidentate chelates
with larger denticities for the purpose of labelling a variety of
radionuclides with the same ligand system in optimized yields.
1
(2; 1.107 g, 6.70 mmol, 48%). H NMR (300 MHz, CDCl3, d):
10.20 (s, aldehyde-H, 1 H), 8.35 (d, py-H, 1 H), 8.15 (d, py-H, 1
H), 8.04 (t, py-H, 1 H), 4.08 (s, CH3, 3 H). 13C NMR (75 MHz,
CDCl3, d): 192.8, 165.0, 152.9, 148.7, 138.6, 129.2, 124.5, 53.5.
HR-MS (ESI+ of M + H+): m/z calcd for C8H8NO3: 166.0504,
found: 166.0501.
3 (Me2dipin). 4-Nitrobenzylamine · HCl (0.457 g, 2.42 mmol)
was stirred in dichloromethane (7 mL). NaOH (1 M, 3.5 mL)
and H2O (2 mL) were added to the suspension; the mixture
was stirred for 30 min until all solids dissolved. The organic
layer was separated from the aqueous layer, which was extracted
with dichloromethane (5 ¥ 5 mL). The combined organic layer
was reduced by rotary evaporation to afford an orange oil. To
this orange oil was added methyl 6-formylpyridine-2-carboxylate
(2; 0.800 g, 4.84 mmol, 2 equiv.) and NaBH(OAc)3 (2.567 g,
12.11 mmol, 5 equiv.), and the mixture was refluxed for 25 h
in 1, 2-dichloroethane (80 mL). The reaction was quenched
with saturated aqueous Na2CO3 (40 mL) and extracted with
dichloromethane (5 ¥ 15 mL). The combined organic layer was
dried over anhydrous MgSO4. The solvent was removed to give
a brown oil, which was recrystallized with 4 : 1 MeOH–DCM.
Brown crystals were collected (0.580 g, 1.29 mmol, 53%). 1H NMR
(300 MHz, CDCl3, d): 8.17 (d, py-H, 2 H), 8.02 (d, py-H, 2 H),
7.85 (t, py-H, 2 H), 7.78 (d, bz-H, 2 H), 7.61 (d, bz-H, 2 H), 4.01 (s,
CH3, 6 H), 3.96 (s, CH2-py, 4 H), 3.83 (s, CH3, 6 H). 13C NMR (75
MHz, CDCl3, d): 165.9, 159.6, 147.7, 147.4, 146.8, 137.7, 129.7,
126., 124.0, 123.8, 60.0, 57.9, 53.1. HR-MS (ESI+ of M + Na+):
m/z calcd for C23H22NaN4O6: 473.1437, found: 473.1433.
Experimental
Materials and methods
All solvents and reagents were used as received. The analytical
thin-layer chromatography (TLC) plates were aluminium-backed
ultrapure silica gel 60, 250 mm; the flash column silica gel
˚
(standard grade, 60 A, 32–63 mm) used was provided by Silicycle.
1H, 13C NMR spectra were recorded on Bruker AV300, AV400
or AV600 instruments; the NMR spectra are expressed on the
ppm scale and are referenced to residual solvent peaks or inter-
nal tetramethylsilane. Electrospray ionization mass spectrometry
(ESI-MS) was recorded on a Micromass LCT instrument at
the Department of Chemistry, University of British Columbia.
IR spectra were collected neat in the solid state on a Thermo
Nicolet 6700 FT-IR spectrometer. 64Cu was obtained as a dilute
HCl solution (MDS Nordion), it was commercially available
from Nordion at the time of the 64Cu experiments. Product
specification reports specific activity of the to be >5000 Ci/g with
< 0.2 micrograms of Cu per mCi. The HPLC system used for
analysis of the 64Cu labelled compounds consisted of a Waters
Alliance HT 2795 separation module equipped with a Raytest
Gabbistar NaI detector and a Waters 996 photodiode array
(PDA) detector. Analysis of 64Cu labelled compounds and their
serum stability was analyzed on a Waters XBridge BEH130 4.6 ¥
150 mm column. [99mTcO4]- was provided by Vancouver Coastal
Health UBC hospital. Reaction control of 99mTc experiments was
performed with TLC (see above), with a mixture of methanol
and dichloromethane (1 : 4) as mobile phase. Radioactive TLCs
were measured using a phosphor imager (Cyclone storage phos-
phor imager with 20 ¥ 25 cm2 phosphor screen, Perkin–Elmer,
Waltham, MA, USA) and analyzed using OptiQuest software.
The [99mTc(H2O)3(CO)3]+ precursor was synthesized using the
Isolink kit generously provided by Covidien, St. Louis, MO.
4-(Aminomethyl)-N-(9-fluorenylmethoxycarbonyl) phenylamine
hydrochloride and biotin-TFP were prepared according to litera-
ture steps.22,26
4 (H2dipin). 2 (0.102 g, 0.227 mmol) was dissolved and stirred
in THF (16 mL). LiOH (22.0 mg, 0.908 mmol, 4 equiv.) was added.
The solution turned light yellow. The mixture was stirred for 2 h,
after which the solvent was removed by rotary evaporation. The
crude mixture was then washed with dichloromethane to yield a
yellow solid (4). 1H NMR (300 MHz, D2O, d): 8.03 (d, py-H, 2 H),
7.68 (t, py-H, 2 H), 7.61 (t, py-H, 2 H), 7.53 (d, bz-H, 2 H), 7.44
(d, bz-H, 2 H), 3.83 (s, CH2-py, 4 H), 3.79 (s, CH2-bz, 2 H). 13C
NMR (75 MHz, D2O, d): 172.6, 157.2, 152.2, 146.2, 146.0, 137.6,
129.6, 125.2, 122.8, 121.6, 60.0, 58.2. IR (neat, cm-1): u(C O) =
1616 cm-1, u(N O) = 1515 cm-1, u(pyridine) = 1587, 1571, 1468,
1436 cm-1. HR-MS (ESI+ of M - H+): m/z calcd for C21H17N4O6:
421.1148, found: 421.1145.
5
(Me2dipiam-Fmoc). 4-(Aminomethyl)-N-(9-fluorenyl
methoxycarbonyl)phenylamine hydrochloride was treated with
saturated Na2CO3 (5 mL) and extracted with DCM (3 ¥ 5 mL).
The combined DCM layer was evaporated to afford the amine as a
white solid. To this amine (0.105 g, 0.305 mmol) was added methyl
6-formylpyridine-2-carboxylate (2; 0.101 g, 0.612 mmol, 2 equiv.)
and NaBH(OAc)3 (0.323 g, 1.52 mmol, 5 equiv.), and the mixture
was refluxed for 15 h in 1, 2-dichloroethane (12 mL). The colour
of the reaction turned from yellow to orange during the reaction.
The reaction was quenched with saturated aqueous Na2CO3
solution (10 mL) and extracted with dichloromethane (4 ¥ 5 mL).
The combined organic layer was dried by rotary evaporation. The
crude product was purified by column chromatography (SiO2, 5%
2 (Methyl 6-formylpyridine-2-carboxylate). 6-Hydroxymethyl
pyridine-2-carboxylic acid methyl ester (1, 2.333 g, 13.95 mmol)
and SeO2 (1.421 g, 12.80 mmol) were refluxed in 1, 4-dioxane
(80 mL) for 2.5 h. The reaction mixture was cooled to room
temperature, and the black solid was filtered off. Dioxane was
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Dalton Trans., 2011, 40, 6253–6259 | 6257
©

MeOH in DCM) as a yellow oil (5; 0.074 g, 0.115 mmol, 38%).
1H NMR (300 MHz, D2O, d): 7.99 (dd, Fmoc-H, 2 H), 7.76–7.83
(m, Fmoc-H, 6 H), 7.60 (d, bz-H, 2 H), 7.41 (t, Fmoc-H, 2 H),
7.29–7.34 (m, Fmoc-H/bz-H, 6 H), 4.55 (d, CO2-CH2, 2 H), 4.27
(t, fmoc-CH, 1 H), 3.99 (s, CH3, 6 H), 3.92 (s, CH2-py, 4 H),
3.65 (s, CH2-bz, 2 H). 13C NMR (75 MHz, D2O, d): 166.0, 160.6,
147.5–120.2, 67.0, 59.9, 58.3, 53.1, 47.3. HR-MS (ESI+ of M +
Na+): m/z calcd for C38H34NaN4O6: 665.2376, found: 665.2384.
[Cu(4)]. 4 (0.005 g, 0.0126 mmol) was stirred in MeOH–
H2O (1 : 1 mL). The pH was adjusted with 0.1 M HCl to 2–3.
Cu(C2H3O2)·H2O (0.0025 g, 0.0126 mmol, in 2 : 1 MeOH–H2O)
was added, and the solution turned clear green. The pH was raised
to 7–8 with 0.1 M NaOH, and a solid precipitated. The mixture
was left to stir for 3 h, and the solvent was removed by rotary
evaporation to give a green solid. Small green plates suitable for
X-ray diffraction were afforded through slow evaporation of a
solution of the complex in a water–methanol mixture (1 : 3). IR
(neat, cm-1): 1639, 1513, 1600, 1571, 1465, 1427. HR-MS (ESI+
of M + Na+): m/z calcd for C21H1663CuN4O6: 506.0264, found:
506.0272. Anal. calcd (found) for C21H16CuN4O6·3NaCl·6H2O: C
32.9 (33.4), H 3.67 (3.7), N 7.3 (6.77).
6 (Me2dipiam). Me2dipiam-Fmoc (5; 0.040 g, 0.0623 mmol)
was stirred in 20% piperidine/DMF (4 mL) for 30 min at room
temperature. A small volume of DCM was added, and the mixture
was extracted with aqueous saturated NaHCO3 solution (3 ¥
5 mL). Basic extraction with NaHCO3 was repeated (3 ¥ 5 mL).
The organic layer was evaporated and then triturated with hexane.
Rotary evaporation to remove hexane afforded yellow solid (6;
[Co(4)]. 4 (0.005 g, 0.0119 mmol) was stirred in MeOH–
H2O (2 : 2 mL). The pH was adjusted with 0.1 M HCl to 2–3.
CoCl2·6H2O (0.003 g, 0.0126 mmol, in 1 : 1 MeOH–H2O) was
added, and the solution turned light pink. The pH was raised
to 7–8 with 0.1 M NaOH, and the colour turned light purple.
The mixture was left to stir for 1 h, after which the solvent was
removed by rotary evaporation to afford a blue solid. IR (neat):
1632, 1518, 1598, 1468, 1436. HR-MS (ESI+ of M + Na+): m/z
calcd for C21H1659CoN4O6: 502.0300, found: 502.0291.
1
0.024 g, 92%). H NMR (300 MHz, CDCl3, d): 7.99 (dd, py-H,
2 H), 7.81 (m, py-H, 4 H), 7.18 (t, bz-H, 2 H), 6.65 (d, bz-H, 2
H), 3.99 (s, CH3, 6 H), 3.92 (s, CH2-py, 4 H), 3.58 (s, CH2-bz, 2
H). 13C NMR (75 MHz, CDCl3, d): 165.1, 160.9, 147.5, 145.8,
137.5, 130.3, 126.9, 126.1, 123.7, 115.2, 59.8, 58.5, 53.1. HR-MS
(ESI+ of M + Na+): m/z calcd for C23H24NaN4O4: 443.1686, found:
443.1695.
[Re(CO)3(3)]Br. 3 (0.044 g, 0.098 mmol) was dissolved
in dichloromethane (5 mL). [Re(CO)3(Br)3][N(Et)4]2 (0.076 g,
0.98 mmol) was dissolved in MeOH (5 mL) and added dropwise
to the ligand solution. The reaction mixture was heated for 18 h at
40 ◦C. Subsequently, the solvent was removed and the crude was
triturated with dichloromethane to remove unreacted ligand and
afford the product as a white solid (0.032 g, 0.043 mmol, 44%) 1H
NMR (300 MHz, CD3OD, d): 8.41 (d, py-H, 2 H), 8.09 (t, py-H,
2 H), 8.01 (d, py-H, 2 H), 7.79 (d, bz-H, 2 H), 7.69 (d, bz-H, 2
H), 5.16–4.58 (m, CH2-py, 4 H), 4.05 (s, CH3, 6 H). 13C NMR (75
MHz, CD3OD, d): 195.7, 191.6, 163.4, 142.7, 140.8, 139.6, 135.0,
131.4, 126.8, 125.2, 124.7, 70.7, 66.9, 64.1, 59.7 IR (neat, cm-1):
2034, 2011, 1895, 1867, 1728, 1370. HR-MS (ESI+ of M+): m/z
calcd for C26H22N4O9Re: 719.0917, found: 719.0906.
7 (Me2dipiam-biotin). Me2dipiam (6; 0.173 g, 0.4 mmol) and
triethylamine (173 mL, 1.245 mmol) were stirred in DMF (6 mL),
biotin-TFP (0.181 g, 0.461 mmol) was added, and the mixture
was stirred overnight at 60 ◦C. The solvent was removed and the
crude was precipitated with THF as a white solid (7; 0.105 g,
1
0.163 mmol, 40%) which was collected by filtration. H NMR
(300 MHz, CD3OD, d): 9.84 (s, 1 H, amide-H), 7.95 (m, py-H,
4 H), 7.81 (d, py-H, 2 H), 7.54 (d, bz-H, 2 H), 7.35 (d, py-H, 2
H), 6.39 (m, 2 H, ureate-H), 4.30 (m, 1 H), 4.15 (m, 1 H), 3.87 (s,
O-CH3, 6 H), 3.77 (s, N-CH2, 4 H), 3.60 (m, N-CH2,4 H), 3.09 (m,
1 H), 2.81 (dd, 2 H), 2.59 (m, 3 H), 2.55 (m, 2 H), 1.51 (m, 6 H).
13C NMR (75 MHz, CD3OD, d): 174.4, 165.3, 162.7, 159.7, 146.7,
137.9, 129.2, 126.2, 123.3, 118.9, 67.0, 61.1, 59.2, 55.4, 52.4, 33.5,
28.2, 28.1, 25.1, 24.5. IR (neat, cm-1): 1690, 1457, 1314. ESI-MS
(ESI+ of M + H+): m/z calcd for C33H39N6O6S: 647.2652, found:
647.2645.
[Cu(8)]. The biotinylated complex was afforded through the
same synthetic procedure as Cu(4) from 7 and Cu(C2H3O2)·H2O as
a light green solid in quantitative yield. HR-MS (ESI+ of M + Na+):
m/z calcd for C31H3263CuN6O6SNa: 702.1398, found: 702.1406. IR
(neat, cm-1): 1684, 1552, 1410.
8
(H2dipiam-biotin). Me2dipiam-biotin (7; 0.008 g,
0.012 mmol) was dissolved in THF–H2O–DCM (1 : 2 : 1).
LiOH (0.002 g, 0.08 mmol) was added and the reaction was
monitored by TLC (20% MeOH in DCM). After the reaction
was found to be complete, the solvent was removed in vacuo to
afford a yellow solid which was redissolved in MeOH and filtered
to remove insoluble impurities. The product was isolated as a
light yellow solid (6.8 mg, 0.011 mmol, 91%). 1H NMR (300
MHz, CD3OD, d): 8.01 (m, 4 H), 7.87 (d, py-H, 2 H), 7.49 (d,
bz-H, 2 H), 7.41 (d, py-H, 2 H), 7.32 (m, bz-H, 2 H), 4.44 (m,
1 H), 4.27 (m, 1 H), 3.73 (s, 4 H, N-CH2), 3.60 (m, N-CH2, 2
H), 3.19 (m, 1 H), 2.88 (dd, 2 H), 2.65 (m, 2 H), 2.13 (m, 2 H),
1.51 (m, 6 H). 13C NMR (75 MHz, CD3OD, d): 183.0, 172.4,
166.3, 158.7, 139.5, 136.5, 131.0, 127.6, 123.8, 121.5, 115.9, 63.4,
61.8, 41.2, 39.1, 30.31, 29.9, 29.7, 27.7. IR (neat, cm-1): 1682,
1567, 1429. ESI-MS (ESI+ of M - 2H+ +3Li+): m/z calcd for
C31H32Li3N6O6S: 623.2264, found: 623.2274. Anal. calcd (found)
for C21H16CuN4O6·HCl·H2O: C 52.8 (52.2), H 4.44 (4.16), N
11.75 (11.43).
[Co(8)]. The biotinylated complex was afforded through the
same synthetic procedure as Co(4) from 7 and CoCl2·6H2O as a
light red solid in quantitative yield. HR-MS (ESI+ of M + Na+):
m/z calcd for C31H3259CoN6O6SNa: 698.1334, found: 698.1350. IR
(neat, cm-1): 1667, 1593, 1403.
[Re(CO)3(7)]Br. 7 (0.005 g, 0.007 mmol) was dissolved in
MeOH (2 mL). [N(Et)4]2[Re(CO)3(Br)3] (0.006 g, 0.007 mmol) was
added as to the ligand solution. The reaction mixture was heated
for 18 h at 60 ◦C. Subsequently, the solvent was removed and
the crude was purified with preparative TLC and eluted off the
silica with 20% MeOH in DCM. The solvent was removed and the
product was afforded as a colourless solid (0.001 g, 0.001 mmol,
14%) 1H NMR (300 MHz, CD3OD, d), 8.09 (m, py-H, 2 H), 7.79
(m, py, 2 H), 7.65 (m, bz-H, 4 H), 5.11–4.94 (m, 4 H, N-CH2),
4.60 (m, N-CH2, 2 H), 4.48 (m, 1 H), 4.30 (m, 1 H), 3.21 (m, 1 H),
6258 | Dalton Trans., 2011, 40, 6253–6259
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©

2.93 (dd, 2 H), 2.69 (m, 2 H), 2.21 (m, 2 H), 1.78–1.41 (m, 6 H)
13C NMR (75 MHz, CD3OD, d): 195.5, 191.8, 177.9, 174.1, 166.9,
166.5, 163.9, 155.0, 142.9, 134.5, 134.5, 126.9, 121.8, 67.1, 63.7,
57.4, 44.3, 41.5, 35.1, 30.2, 29.9, 26.9, 26.4, 25.9 IR (neat, cm-1):
2034, 2012, 18.98, 1872, 1722, 1698. HR-MS (ESI+ of M+): m/z
calcd for C36H38N6O9SRe: 915.1951, found: 915.1964.
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64Cu labelling and stability. A solution of 64Cu (non-carrier
added, 0.4–0.7 mCi, in 50 mL) was added to a 10-5 M solution
(pH 5, sodium acetate buffer) of ligand. The reaction mixture was
analyzed by HPLC after 10 min at room temperature. To evaluate
complex stability, an aliquot (100 mL) of the reaction was added to
a solution of mouse serum (pH 5 buffer, 0.1 mM glycine, 900 mL).
The mixture was incubated at 37 ◦C and analyzed by HPLC
after 1 h. The following specific activities were measured: 0.066
mCi/nmol (H2dipin) and 0.037 mCi/nmol (H2dipiam–biotin).
99mTc labelling and stability. The organometallic precursor
[
99mTc(H2O)3(CO)3]+ was prepared from a saline solution of
Na[99mTcO4] (1 mL, 100 MBq) using the Isolink kit. A solution
of Na[99mTcO4] (1 mL) was added to the Isolink kit, and the
vial was heated to reflux for 40 min. Upon cooling, 0.1 M HCl
solution (1 mL) was added to adjust the pH to approximately 7.
[
99mTc(H2O)3(CO)3]+ (10 MBq, in 0.2 mL) was added to a 10-4
M
solution of ligand in a sealed vial and heated to reflux for 40 min.
TLC’s were analyzed with a 4 : 1 mixture of dichloromethane
and methanol as mobile phase. To evaluate complex stability, an
aliquot (0.5 mL) of 99mTc-complex was challenged with a 0.1 M
solution of cysteine and histidine (0.5 mL). The mixture was
incubated at 37 ◦C and analyzed by TLC after 24 h.
Acknowledgements
We acknowledge UBC for a 4 Year Fellowship (E. B.), as well
as Nordion (Canada) and the Natural Sciences and Engineering
Research Council (NSERC) for grant support (CRD).
24 C. Platas-Iglesias, M. Mato-Iglesias, K. Djanashvili, R. N. Muller, L.
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This journal is
The Royal Society of Chemistry 2011
Dalton Trans., 2011, 40, 6253–6259 | 6259
©