Rhenium and technetium tricarbonyl complexes of 1,4-Substituted pyridyl-1,2,3-triazole bidentate 'click' ligands conjugated to a targeting RGD peptide

Article abstract of DOI:10.1002/jlcr.3169

New 1,4-substituted pyridyl-1,2,3-triazole ligands with pendent phenyl isothiocyanate functional groups linked to the heterocycle through a short methylene or longer polyethylene glycol spacers were prepared and conjugated to a peptide containing the arginine-glycine-aspartic acid peptide motif. Rhenium and technetium carbonyl complexes, [M(CO)₃L^x(py)] ⁺ (where M = Re^I or ^99mTc^I; L ^x = 1,4-substituted pyridyl-1,2,3-triazole ligands and py = pyridine) were prepared. One rhenium complex has been characterized by X-ray crystallography, and the luminescent properties of [M(CO)₃L ^x(py)]⁺ are reported.

Full text of DOI:10.1002/jlcr.3169

Special Issue Article
Received 9 August 2013,
Accepted 29 October 2013
Published online 11 December 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3169
Rhenium and technetium tricarbonyl
complexes of 1,4-Substituted pyridyl-1,2,
3-triazole bidentate ‘click’ ligands
conjugated to a targeting RGD peptide†‡
Timothy U. Connell,a David J. Hayne,a Uwe Ackermann,b
b
a
a
*
Henri J. Tochon-Danguy, Jonathan M. White, and Paul S. Donnelly
New 1,4-substituted pyridyl-1,2,3-triazole ligands with pendent phenyl isothiocyanate functional groups linked to the
heterocycle through a short methylene or longer polyethylene glycol spacers were prepared and conjugated to a peptide
containing the arginine–glycine–aspartic acid peptide motif. Rhenium and technetium carbonyl complexes, [M(CO)3Lx(py)]+
(where M = ReI or 99mTcI; Lx = 1,4-substituted pyridyl-1,2,3-triazole ligands and py = pyridine) were prepared. One rhenium
complex has been characterized by X-ray crystallography, and the luminescent properties of [M(CO)3Lx(py)]+ are reported.
Keywords: rhenium; technetium; click chemistry; 1,2,3-triazole bidentate ligands; RGD peptide; luminescent Re(CO)3 complexes
to rapidly form complexes of sufficient stability for use in vivo.
The three aqua ligands can be substituted for one bidentate ligand
Introduction
The decay characteristics of the γ-emitting radioactive isotope
and one monodentate ligand to form mixed ligand complexes,
99mTc and its availability from commercial generators have led
to the isotope being the most commonly used radionuclide for
diagnostic imaging using the technique of single photon
computed tomography (SPECT).1,2 For most SPECT imaging
applications, the 99mTc isotope is incorporated into a coor-
dination complex where the molecular characteristics dictate
the biodistribution in vivo. Further selectivity and specific
imaging of molecular interactions associated with disease prog-
ression is possible by tethering the 99mTc complex to targeting
molecules. Technetium is positioned in the middle of the
transition metal series and is capable of existing in a wide range
of oxidation states. Recent innovations in the aqueous chemistry
of low valent technetium(I) carbonyl complexes have led to kit
formulations for the preparation of the stable fac-[TcI(CO)3(OH2)3]+
core where the three facially coordinated carbonyl ligands stabilize
the TcI ion. The ‘carbonyl core’ approach exploits the kinetic stability
of the d6 TcI metal tricarbonyl core whilst manipulating the relatively
labile water ligands to attach pertinent biomolecules.3–5 A variety of
mono-, bi- and tri-dentate ligands can react with the tricarbonyl core,
displacing the substitutionally labile aqua ligands. The third row
congener to technetium, rhenium, is also of interest as there are
two β-emitting isotopes 186/188Re that have the potential to be of
and this has been referred to as the [2+ 1] mixed ligand concept.8
In this manuscript, we use CuI-catalysed azide–alkyne cyclo-
addition (CuAAC),
a variant of the Huisgen azide–alkyne
cycloaddition, to prepare 1,4-substituted pyridyl-1,2,3-triazole
bidentate ligands, Lx. The ligands have been elaborated with a
terminal isothiocyanate functional group through both an
aromatic and polyethyleneglycol spacer. The isothiocyanate
functional group was used to conjugate a targeting peptide
with an arginine–glycine–aspartate (RGD) motif to the pyridyl-
1,2,3-triazole bidentate ligands. Cyclic peptides with the
sequence cyclic-(RGDxK) (where x = DPhe(f) or DTyr(y)) bind
with high affinity and specificity to the αvβ3 integrin receptor
that is overexpressed in activated endothelial cells of cancer
neovasculature and some types of tumours. Diagnostic imaging
aSchool of Chemistry, Bio21 Molecular Science and Biotechnology Institute,
University of Melbourne, Melbourne, 3010, Australia
bCentre for PET, Austin Health, Melbourne, 3084, Australia
*Correspondence to: Paul S. Donnelly, School of Chemistry, Bio21 Molecular Science
use in radiotherapeutic applications.6,7 The similar ionic radii of and Biotechnology Institute, University of Melbourne, Melbourne 3010, Australia.
E-mail: pauld@unimelb.edu.au
technetium and rhenium often leads to their carbonyl complexes
† Additional supporting information may be found in the online version of this
being essentially isostructural, and the two elements offer the
potential of being used in concert for combined diagnostic and article at the publisher’s web-site.
therapeutic applications.
‡ This article is published in the Journal of Labelled Compounds and
Radiopharmaceuticals as a special issue on ‘Current Developments in PET and
SPECT Imaging‘, edited by Jonathan R. Dilworth, University of Oxford and Sofia
Detailed and thorough studies of substitution reactions of
[M(H2O)3(CO)3]+ (where M = Tc or Re) have identified heterocyclic
ligands such as imidazole or pyridine derivatives as ideally suited I. Pascu, University of Bath.
J. Label Compd. Radiopharm 2014, 57 262–269
Copyright © 2013 John Wiley & Sons, Ltd.

T. U. Connell et al.
of integrin expression is of interest in characterizing tumour-
associated angiogenesis.9–20
Results and discussion
Ligand synthesis and functionalization
Incorporation of a polyethylene glycol linker into the pyridyl-
1,2,3-triazole framework was achieved via a CuAAC reaction
with 1-azido,13-(Boc-amino)-4,7,10-trioxatridecane (3) and 2-
ethynylpyridine to give L2a. The azido-polyether was prepared
by selective protection of a single amine site of 1,13-diamino-
4,7,10-trioxatridecane (1) with di-tert-butyl dicarbonate follo-
wed by conversion of the remaining amine to an azido
functional group with triflic azide to give 3 (Scheme 1B).
Deprotection of the amine in L2a and addition of an excess of
p-phenylene diisothiocyanate allowed isolation of L2b furnished
with a phenylisothiocyante functional group poised for biocon-
jugation reactions with amines (Scheme 2). Conjugation of L2b
to the cyclic pentapeptide cRGDfK (cyclic Arg-Gly-Asp-D-Phe-Lys)
was achieved in the presence of diisopropylethylamine followed
by purification by semi-preparative HPLC (Scheme 2, Figure 1).
A pyridyl-1,2,3-triazole ligand featuring a shorter linker to the
aromatic isothiocyanate functional group was also prepared (L3).
The synthesis of L3 involved the in situ synthesis of the requisite
para-nitro benzyl azide from the reaction of the corresponding
bromide substituted precursor 4 with sodium azide,21 followed
by CuAAC mediated triazole formation with 2-ethynylpyridine to
give 5 (Scheme 3). The isothiocyanate, L3 was prepared by
reduction of the –NO2 functional group to the corresponding
amine with SnII chloride followed by conversion of the amine to
Scheme 2. Synthesis of cyclic pentapeptide cRGDFK conjugate L2b-cRGDfK.
Reagents and Conditions: (i) (a) TFA, DCM, 25°C, 2 h; (b) p-phenylene diisothiocyanate,
DCM, 25°C, 14h; and (ii) cRGDfK, DIPEA, DMF, 25°C, 12 h.
an isothiocynate functional group with thiophosgene to give L3.
The reaction of L3 with cRGDfK allowed isolation of L3-cRGDfK
(Scheme 3). Formation of the isothiocyanate via the dithiocar-
bamate intermediate was attempted but proved to be unsu-
ccessful, most likely due to the electron withdrawing effect of
the triazole ring. The novel ligands L2a, L2b and L3 were charac-
terized by 1H-nuclear magnetic resonance (NMR) and 13C-NMR
along with HR-ESMS.
Synthesis and characterization of rhenium(I) tricarbonyl
complexes with 1,4-substituted pyridyl-1,2,3-triazole
bidentate ligands
Ligands containing triazole heterocycles have been previously
exploited in the coordination chemistry of rhenium carbonyl
complexes, both as bidentate and monodentate ligands.22–28
Scheme 1. Synthesis of 4-(2-pyridyl)-1,2,3 triazole ligands. Reagents and conditions:
(i) CuSO4, sodium ascorbate, DMSO/H2O (2:1 v/v), 25°C, 24h; (ii) Boc2O, dioxane, 25°
C, 20 h; and (iii) TfN3, K2CO3, CuSO4, DCM/H2O (1:1 v/v), 25°C, 48h.
Figure 1. HPLC trace of the ligand conjugate L2b-cRGDfK. Inset shows HR-ESMS
spectra of fraction with experimental and calculated values.
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T. U. Connell et al.
Scheme 4. Synthesis of rhenium(I) complexes. Reagents and Conditions: (i) L1,
MEOH, 65°C, 16 h; (ii) L2a, toluene, 110°C, 30 min; and (iii) AgOTf, pyridine, DCM,
rt, 2 h➔ 40°C, 16 h.
dichloromethane and pentane. The X-ray structure (Figure 3)
reveals a six coordinate rhenium(I) complex with the metal ion
in a distorted octahedral environment. The three carbonyl ligands
are in a facial arrangement about the ReI cation with one apical
position occupied by a coordinated pyridine. The coordination
sphere is completed by L1, which acts as a bidentate ligand. The
Re-N(pyridyl) bond distances (≈2.20 Å) are slightly longer than the
Re-N(triaozlyl) distance (≈2.139 Å) but consistent with similar
Scheme 3. Synthesis of isothiocyanato functionalized 4-(2-pyridyl)-1,2,3 triazole
ligand and its cyclic cRGDfK conjugate. Reagents and Conditions: (i) NaN3, Na2CO3,
CuSO4, sodium ascorbate, DMF/H2O (4:1 v/v), 25°C, 20 h; (ii) (a) SnCl2, EtOH, 78°C,
16h; (b) thiophosgene, DCM, 0°C, 30 min; and (iii) cRGDfK, DIPEA, DMF, 25°C, 12h.
The mild reaction conditions required to prepare these ligands structures.22,23,25,29 The bite angle for the bidentate ligand
has stimulated the synthesis of numerous monofunctionalized (74.43°) is similar to the angle found in the previously published
bidentate derivatives, but there are few examples that possess structure [Re(CO)3L1Cl] (74.5°).22 The C≡O bond length trans to
biomolecules or functional groups suitable for bioconjugation the triazolyl-ring is 1.139(4) compared with those trans to the
to molecules that could engender biological selectivity.22,29 pyridyl or pyridine ring, 1.149(4) and 1.153(4), respectively.
Related complexes have been investigated as multi-modality Synthesis of the complex [Re(CO)3L3Br] was successful, but
imaging agents [infrared (IR) combined with fluorescence conversion to the [Re(CO)3L3(py)](OTf) was difficult, and it is
detection].30
suspected that intramolecular coordination of the isothiocyanate
The synthesis of the [2 + 1]-type rhenium carbonyl complexes functional group to the rhenium centre upon halide abstraction
was achieved by the reaction of ligand L1 with [Re(CO)3(py)3]OTf complicates the reaction.
(where py = pyridine, Scheme 4A).31 The [Re(CO)3Lx(py)]
The photophysical properties of complexes of the type [fac-Re
complexes can also be prepared by bromide abstraction of the (CO3)N^NX] (where N^N) is a bidentate diimine ligand such as
[Re(CO)3LxBr] complex with silver triflate in the presence of bipyridine or phenathroline and ’X’ is halide anion are of interest
pyridine (Scheme 4).22,25
in the development of photosensitisers for energy conversion
[Re(CO)3LxBr] can be prepared by the reaction of [Re(CO)5Br] and as chemical sensors.32–34 The emission spectra of solutions
with the bidentate pyridyl-1,2,3-triazole ligand in toluene. Each of [Re(CO)3L1(py)]OTf and [Re(CO)3L2a(py)]OTf in acetonitrile
of the rhenium complexes was analysed by electrospray mass show a blue shift compared with [Re(CO)3(bpy)Br] (Table 1).35
spectrometry, and signals for the monocations were the major Interestingly, [Re(CO)3L1(py)]OTf shows a blue shift in the
peak in each spectrum with the expected isotope pattern: for emission maxima when compared with the halide analogue
example, m/z 799.2439 for [Re(CO)3L2a(py)]+ ([C30H40N6O8Re]+ [Re(CO)3L1Cl] (Figure 4).22,25
requires 799.2465). The H-NMR and 13C-NMR spectra of these
It is also noteworthy that previous research has found that
1
compounds were as expected. Coordination of the ligand to different functional groups on the triazole have little effect on
the ReI cation results in a downfield shift of 1H-NMR signals emission properties, yet [Re(CO)3L2a(py)]OTf is significantly red
attributed to the pyridyl-rings and triazolyl-rings when com- shifted compared with the benzyl derivative L1. Luminescent
pared with the ‘free’ ligands. This downfield shift is particularly quantum yields were calculated relative to [Re(CO)3(bpy)Br],35
evident in the triazole proton: it shifts from 8.07 ppm in the free and were found to be similar to other rhenium complexes
ligand L1 to 9.45 ppm in the complex [Re(CO)3L1(py)]OTf containing 4-(2-pyridyl)-1,2,3 triazole ligands.25 The phospho-
(Figure 2).
rescent properties of [Re(CO)3LX(py)] and [Re(CO)3LX(Br)] may
Single crystals of [Re(CO)3L1(py)]OTf suitable for analysis by be of use in tracking the cellular metabolism and binding
single crystal X-ray crystallography were grown by diffusion of affinity of the new complexes for αvβ3 integrin receptor using
vapours between a solution of the complex dissolved in confocal microscopy.36–40
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T. U. Connell et al.
Figure 2. Partial 1H-NMR spectra (500 MHz, CDCl3, 298 K) of the ligand L1 (top) and the complex [Re(CO)3L1(py)]OTf.
Figure 3. An ORTEP representation of the cation [Re(CO)3L1(py)]+. Ellipsoids are
shown at the 20% probability level. The hydrogen atoms have been removed for
clarity.
Table 1. Emission maxima and quantum yield measure-
ments of rhenium complexes
λmax (nm)
Φ
[Re(CO)3(bpy)Br]
597
496
555
7.1 × 10-3a
3.1 × 10-2
1.9 × 10-2
[Re(CO)3L1(py)]OTf
[Re(CO)3L2a(py)]OTf
aAs reported in literature, see footnote 35.
Figure 4. Absorbance (A) and normalized emission (B) spectra (irradiated at
350 nm) of 20 μM solutions of [Re(CO)3L1(py)]OTf (blue), [Re(CO)3L2a(py)]OTf (black)
and [Re(CO)3(bpy)Br] (red).
Synthesis of [99mTc(CO)3L1-3X] complexes
Radiolabelling was carried out using
[
99mTc(CO)3(H2O)3]+
involved adding pyridine (5 μL of a 5% aqueous solution) and
heating for a further 60 min. The synthesis of the desired
complex, [99mTc(CO)3Lx(py)], was then confirmed by compa-
rative RP-HPLC with the corresponding rhenium analogue. The
triazole-based bidentate ligand shows efficient labelling, with
radiopurity of a crude radiochemical yield >95% for [99mTc(CO)
3L1X] (Tr = 12.6 min). Following addition of pyridine, two major
prepared from [99mTcO4- ] (1000 MBq, in 0.9% saline solution
(1 mL)) using an Isolink kitTM. The synthesis of complexes
containing both a triazole-based bidentate ligand and pyridine
as monodentate ligand was carried out in a two-step process.
First, the bidentate ligand was reacted with [99mTc(CO)3(H2O)3]+
for 30 min at 75°C (pH 7.5). The second step of the synthesis
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T. U. Connell et al.
products are evident. The first peak in the chromatogram is most coordination of pyridine was observed, with retention times
likely due to unreacted [99mTc(CO)3L1X] whilst the second peak being slightly increased. The ligand containing cyclic RGDfK
(Tr = 13.2min) is consistent with the retention time for the rhenium labelled with a radio purity of >95%, but a second peak with a
analogue, [Re(CO)3L1(py)] (Tr = 13 min). The slight variation in longer retention time was not detected after reacting with
retention time is due to the equipment configuration, with the pyridine (Figure 6). This is probably because coordination of a
injected sample flowing through the ultraviolet (UV) absorbance pyridine to the complex to give [Re(CO)3L2b-cRGDfK(py)]+ does
detector before passing the γ counter (Figure 5).
not affect lipophilicity sufficiently to give a different retention
Ligand L2a also labelled with high effiency, giving [99mTc(CO) time compared with [Re(CO)3L2b-cRGDfK(X)] for the RP-HPLC
3L2aX] in approximately 85% radiochemical purity, with the conditions used. Attempts to radiolabel L3 and L3-cRGDfk with
addition of pyridine giving [99mTc(CO)3L2a(py)] in approximately
[
99mTc(CO)3]+ resulted in mixtures with several products
65% radiochemical purity. A slight increase in lipophilicity upon observed by RP-HPLC. This is consistent with the unsuccessful
attempts to synthesize the corresponding rhenium complexes.
Conclusion
New 1,4-substituted pyridyl-1,2,3-triazole ligands and complexes
of general formula [Re(CO)3Lx(py)]+ have been prepared.
Complexes [Re(CO)3L1(py)]+ and [Re(CO)3L2a(py)]+ are lumine-
scent with quantum yields comparable with [Re(CO)3(bipy)Br]+.
New 1,4-substituted pyridyl-1,2,3-triazole ligands with pendent
phenyl isothiocyanate functional groups linked to the heterocycle
through a short methylene linker (L3) or a longer polyethylene
glycol spacers (L2b) were prepared and conjugated to a cyclic-
(RGDfK) peptide that is known to have high affinity and specificity
for αvβ3 integrin receptors. Ligand L1 was radiolabelled with
[
99mTc(CO)3]+ to give the [2+ 1] complexes [99mTc(CO)3L1X]+
(where X = solvent) in high-radiochemical yield. There was some
evidence that addition of pyridine to these mixtures resulted in
exchange of the [+1] ligand to pyridine. Of the two ligands
conjugated to cRGDfK, the ligand with longer polyethylene glycol
spacer (L2b-cRGDfK) demonstrated superior radiolabeling with
[
99mTc(CO)3]+ to give [99mTc(CO)3L2bX] (where X = solvent or
pyridine). Future studies will focus on the use of these new
bidentate 1,4-substituted pyridyl-1,2,3-triazole ligands with
isonitrile co-ligands in place of pyridine.41,42
Experimental
Instrumentation
Nuclear magnetic resonance spectra were acquired on a Varian FT-NMR
500 spectrometer (Varian, California, USA). 1H-NMR spectra were
acquired at 500 MHz, and 13C{1H}-NMR spectra were acquired at
125.7 MHz. All NMR spectra were recorded at 25°C. All chemical shifts
were referenced to residual solvent peaks and are quoted in ppm relative
to TMS. ESI-MS spectra were recorded on an Agilent 6510 ESI-TOF LC/MS
Mass Spectrometer (Agilent, California, USA). HPLC traces were acquired
using an Agilent 1200 Series HPLC system (Agilent, California, USA) with
Figure 5. From top to bottom radio-HPLC of [Re(CO)3L1(py)]+, radio-HPLC of
99mTc(CO)3L1X] (X = axial ligand) and radio-HPLC of [99mTc(CO)3L1(py)]+.
[
a
SGE Analytical Science ProteCol C18 HPH125 120 Å column
(4.6 × 150 mm, 5 μm), a gradient elution of H2O–CH3CN/0.1% trifluo-
roacetic acid, 0–100% CH3CN, a 1 mL min-1 flow rate over 25 min and
were monitored at λ = 220, 254, 280 and 320 nm. Semi-preparative HPLC
purifications were performed using an Agilent 1200 Series HPLC system.
Solvent gradients and column specifications are described in the
succeeding texts. An automated Agilent 1200 fraction collector collected
2.5-mL fractions, and fraction collection was time-based. Each fraction
was analysed using MS and analytical HPLC. Reverse phase HPLC method
employed a Phenomenex Luna 5u C18(2) 100A 150 × 21.20 mm column
with a flow rate of 5 mL min-1. The gradient mobile phase started with
100% solvent A (0.1% trifluoroacetic acid in water) at 5 min to 80%
solvent B (0.1% trifluoroacetic acid in acetonitrile) at 85 min. Analytical
radio-HPLC was performed using a Shimadzu 10AVP UV–vis detector
(Shimadzu, Kyoto, Japan), 2 LC-10ATVP solvent delivery systems (for
solvent A & B), a Nacalai Tesque Cosomosil 5C18-AR Waters column
(4.6 mm I.D. × 150 mm) (Nacalai Tesque, Kyoto, Japan). The mobile phase
Figure 6. Radio-HPLC of [99mTc(CO)3(L2b-cRGDfK)X].
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T. U. Connell et al.
2-(1-(benzyl)-1H-1,2,3-triazol-4-yl)pyridine (L1)
was a gradient consisting of 5% solvent B at t = 0 to 100% solvent B after
20 min, where solvent A = MilliQ water containing 0.1% TFA and solvent
B = acetonitrile containing 0.1% TFA. All runs were conducted at a
constant total flow rate of 1 mL min-1 and were monitored at
λ = 254 nm. Absorbance spectra were obtained on a Shimadzu UV-1650
PC spectrophotometer, and emission spectra were obtained on a Varian
CARY Eclipse fluorescence spectrophotometer. Both measurements were
performed in capped quartz cuvettes. IR spectra were obtained on a
Perkin–Elmer Spectrum One FTIR spectrometer, with a zinc selenide/
diamond universal ATR 60 sampling accessory, as a thin film.
A
mixture of benzyl azide (0.350 g, 2.63 mmol), 2-ethynylpyridine
(0.25 mL, 2.47 mmol), CuSO4.5H2O (12 mg, 2 mol%) and sodium ascorbate
(100 mg, 20 mol%) were stirred at room temperature overnight and then
quenched with water (100 mL). The precipitate was collected by filtration
then redissolved in EtOAc (100 mL) and washed with EDTA/NH4OH
solution (1M, 50 mL) and brine (50 mL). The organic layer was separated
and dried (MgSO4) then concentrated under reduced pressure followed
by drying in vacuo to yield an off white powder of L1 (0.365 g, 62%). 1H-
NMR (CDCl3, 500 MHz): δ 5.58 (2H, s, C8H2), 7.20 (1H, dd, J = 6.9, 5.2 Hz,
C2H), 7.32–7.40 (5H, m, phenyl-C10-C12H), 7.76 (1H, td, 7.7, 1.7 Hz, C3H),
8.04 (1H, s, C7H), 8.17 (1H, d, J = 7.9 Hz, C1H), 8.53 (1H, d, J = 3.0 Hz, C4H).
1-amino,13-(Boc-amino)-4,7,10-trioxatridecane (2)
This compound was synthesized by modification of a previously reported
procedure.43 To a solution of 1 (16.2 g, 73.6mmol) in 1,4-dioxane (120 mL),
a solution of Boc2O (2 g, 9.2 mmol), in 1,4-dioxane (50 mL) was added at
room temperature over 14 h. The mixture was stirred at room temperature
for a further 48 h then the solvent was removed by evaporation under
reduced pressure and the residue was dissolved in H2O (50mL). This mixture
was extracted with dichloromethane (4 × 50 mL), and the combined organic
phases washed with brine solution (4 × 50 mL). This extraction procedure
was repeated. The combined organic extracts were dried (MgSO4) and
filtered before the solvent was removed under reduced pressure to yield a
slightly yellow oil (2.232 g, 76%). The doubly protected amine can be
separated by silica gel chromatography using a gradient of 100%
dichloromethane ➔ 10% MEOH, 2% aq. ammonia, 88% dichloromethane.
1H-NMR (CDCl3, 500MHz): δ 1.40 (9H, s, t-butyl-H), 1.72 (4H, m, NH2/NHBoc-
CH2-CH2), 2.13 (s, 3H, NH2), 2.78 (2H, t, J=6.7Hz, NH2-CH2), 3.18 (2H, d,
J= 6.1 Hz, NHBoc-CH2), 3.49–3.62 (12H, m, O-CH2), 5.13 (1H, s, NHBoc).
2-(1-(13-(Boc-amino)-4,7,10-trioxatridecyl)-1H-1,2,3-triazol-4-yl)
pyridine (L2a)
A
mixture of 3 (0.343g, 0.990 mmol), 2-ethynylpyridine (0.1mL,
0.989 mmol), CuSO4.5H2O (5mg, 2mol%) and sodium ascorbate (40mg,
20 mol%) were stirred at room temperature for 4days and then quenched
with water (10mL). The mixture was extracted with dichloromethane
(3× 15mL), and the combined organic extracts were washed with brine
solution, dried (MgSO4) and concentrated under reduced pressure. The
crude oil was purified by silica gel chromatography using EtOAc as the
eluent (compound was loaded as a dichloromethane solution) to yield a
light yellow oil of L2a (0.3864g, 87%). 1H-NMR (CDCl3, 500 MHz): δ 1.40
(9H, s, t-butyl-H), 1.72 (2H, q, J = 6.2Hz, C16-H2), 2.20 (2H, q, J= 6.1Hz, C9-
H2), 3.19 (2H, q, J= 5.18Hz, C17-H2), 3.47 (2H, t, J = 5.8Hz, C10-H2), 3.51 (2H,
t, J= 6.0Hz, C15-H2), 3.56–3.60 (4H, m, C11-C14-H2), 3.61–3.64 (4H, m, C11
14-H2), 4.53 (2H, t, J= 6.8Hz, C8-H2), 5.01 (1H, s, NH), 7.20 (1H, ddd,
-
C
J = 7.5, 4.9, 1.2Hz, C2-H), 7.75 (1H, td, J = 7.7, 1.8Hz, C3-H), 8.15 (1H, dt,
J = 7.9, 1.0Hz, C4-H), 8.16 (1H, s, C7-H), 8.55 (1H, ddd, J = 4.9, 1.8, 0.9Hz,
C1-H). 13C-NMR (CDCl3, 125 MHz): δ 28.53 (C20), 29.77 (C16), 30.38 (C9),
38.65 (C17), 47.46 (C8), 67.22 (C10), 69.66 (C15), 70.34 (C14), 70.52 (C11),
70.66, 70.70 (C12/C13, could not be resolved), 78.97 (C19), 120.29 (C4),
122.60 (C7), 122.85 (C2), 136.95 (C3), 148.34 (C6), 149.47 (C1), 150.52 (C5),
156.132 (C18). HRMS m/z 472.2547 ([M + Na]+ requires 472.2536), m/z
450.2715 ([M + H]+ requires 450.2716).
1-azido,13-(Boc-amino)-4,7,10-trioxatridecane (3)
This compound was synthesized by modification to a previously reported
procedure.44 To a mixture of sodium azide (2.44 g, 37.5 mmol) in water
(10 mL) and dichloromethane (25 mL) at 0°C was added Tf2O (1.26 mL,
7.49 mmol) dropwise. The mixture was allowed to warm to room
temperature and stirred vigorously for 2 h. The organic layer was
separated, and the aqueous layer was further extracted with
dichloromethane (2 × 10 mL). The combined organic extracts were
washed with saturated aqueous Na2CO3 (15 mL).
2-(1-(13-(3-(4-isothiocyanato-phenyl)-thioureido)-4,7,10-
trioxatridecyl)-1H-1,2,3-triazol-4-yl)pyridine (L2b
)
The previously outlined solution was added dropwise to 2 (1.2034 g,
3.76 mmol), K2CO3 (1.04 g, 7.52 mmol) and CuSO4.5H2O (cat.) in water
(20 mL) and MEOH (30 mL). The mixture was stirred vigorously for 48 h
to ensure decomposition of triflic azide. The organic layer was separated
and washed with water (2 × 10 mL). The combined aqueous extracts were
back extracted with dichloromethane (3 × 20 mL). The combined organic
extracts were dried (MgSO4) and concentrated under reduced pressure.
The crude oil was purified by silica gel chromatography using a gradient
of 100% dichloromethane ➔ 6% MEOH, 94% dichloromethane to yield a
slightly yellow oil (1.1635 g, 89%). 1H-NMR (CDCl3, 500 MHz): δ 1.44 (9H, s,
t-butyl-H), 1.76 (2H, quintet, J = 6.2 Hz, C9-H2), 1.85 (2H, quintet, J = 6.4 Hz,
C2-H2), 3.23 (2H, q, J = 5.9 Hz, C10-H2), 3.39 (2H, t, J = 6.7 Hz, C1-H2), 3.54
(4H, 1, J = 5.9 Hz, C3/C8-H2), 3.58–3.66 (8H, m, C4-C7-H2), 4.95 (1H, s, NH).
13C-NMR (CDCl3, 125 MHz): δ 28.61 (C13), 29.30 (C2), 29.80 (C9), 38.76
(C10), 48.62 (C1), 68.02 (C3), 69.80 (C8), 70.41, 70.53, 70.74, 70.76 (C4-C7,
could not be resolved), 79.05 (C12), 156.16 (C11). For molecular labelling
diagrams for this and all future compounds, see Supporting Information.
HRMS (ESI) m/z 369.2111 ([M + Na]+ requires 369.2114), m/z 364.2606
([M+ NH4]+ requires 364.2560), m/z 347.2433 ([M + H]+ requires 347.2294).
To a solution of L2a (156 mg, 346 μmol) in dichloromethane (10 mL) was
added trifluoroacetic acid (1mL), and the solution was stirred at room
temperature for 1.5hours. The solvent was evaporated under reduced
pressure, and the residue was redissolved in dichloromethane (20mL),
washed with saturated aqueous Na2CO3 (20mL), dried (Na2SO4) and
concentrated under reduced pressure. The concentrated solution was
added dropwise to p-phenylene diisothiocyanate (516mg, 2.68mmol)
and triethylamine (192μL, 1.38 mmol) in dichloromethane (20mL) and
then stirred at room temperature for 16 h. Solution was concentrated
under reduce pressure, and the crude oil was purified by silica gel
chromatography using a gradient of 100% EtOAc ➔ 4% MEOH, 96% EtOAc
to yield a slightly yellow oil of L2b (147 mg, 78%). 1H-NMR (CDCl3, 500 MHz):
δ 1.88 (2H, dt, J= 11.4, 5.7Hz, C18-H2), 2.17 (2H, quintet, J= 6.3Hz, C9-H2),
3.43 (2H, t, H = 5.9Hz, C10-H2), 3.45–3.58 (8H, m, C11-C14-H2), 3.61 (2H, t,
J = 5.4Hz, C15-H2), 3.78 (2H, br s, C17-H2), 4.51 (2H, t, J= 6.7Hz, C8-H2),
7.17–7.20 (2H, m, C21-H), 7.24 (1H, ddd, J = 7.5, 4.9, 1.2Hz, C2-H), 7.34–7.35
(2H, m, C20-H), 7.78 (1H, td, J = 7.7, 1.8Hz, C3-H), 8.13 (1H, dt, J= 7.9,
1.0 Hz, C4-H), 8.19 (1H, s, C7-H), 8.30 (1H, s, C = SNH-phenyl), 8.57 (1H, ddd,
J = 4.9, 1.7, 0.9Hz, C1-H). 13C-NMR (CDCl3, 125 MHz): δ 28.21 (C16), 30.26
(C9), 45.12 (C17), 47.44 (C8), 67.35 (C10), 68.83, 70.19, 70.28, 60.51 (C11-C14
could not resolve), 70.79 (broad shoulder, C15), 120.45 (C4), 122.61 (C7),
123.09 (C2), 125.06 (C20), 126.72 (C21/C22), 128.25 (C19), 135.93 (C23),
137.16 (C3), 148.42 (C6), 149.54 (C1), 150.35 (C5), 180.77 (C18). HRMS m/z
542.1996 ([M + H]+ requires 542.2008).
General procedure for CuAAC ‘click’ reactions
General Procedure: To a solution of the azide (1 eq.) in a 2:1 mixture of
DMSO/H2O was added the alkyne (1.1 eq), CuSO4.5H2O (1 mol%), sodium
ascorbate (10 mol%) with or without accelerating ligand, tris[(1-benzyl-
1H-1,2,3-triazol-4-yl)methyl]amine (1 mol%). The solution was stirred at
room temperature and quenched with water (10–30 mL) when
2-(1-(4-nitro-benzyl)-1H-1,2,3-triazol-4-yl)pyridine (5)
determined to be finished by thin-layer chromatography. The product This compound was synthesized by modification of a previously reported
was then isolated by filtration or extraction with an organic solvent.
procedure.21 To a stirred solution of 2-ethynylpyridine (0.62 g, 6.0 mmol)
J. Label Compd. Radiopharm 2014, 57 262–269
Copyright © 2013 John Wiley & Sons, Ltd. www.jlcr.org

T. U. Connell et al.
[Re(CO)3L2aBr]
in a 4:1 mixture of DMF/H2O (15 mL) was added NaN3 (0.43 g, 6.6 mmol),
Na2CO3 (0.63 g, 5.9 mmol), CuSO4.5H2O (0.30 g, 1.2 mmol) and ascorbic
acid (1.19 g, 6.0 mmol). To this was added 4 (1.08, 6.3 mmol), and the
mixture was stirred at room temperature for 20 h. This mixture was
added to an aqueous EDTA/NH4OH solution (100 ml, 1 M) and extracted
with EtOAc (4 × 75 mL). The combined organic extracts were washed with
brine (50 mL), dried (MgSO4) and concentrated under reduced pressure,
then dried in vacuo to yield a brown precipitate (1.30 g, 77%), which
was used without further purification. 1H-NMR (CDCl3, 500 MHz): δ 5.71
(2H, s, triazole-CH2-phenyl), 7.24 (1H, dd, J = 7.5, 4.9, pyridyl-C2H), 7.46
(2H, d, J = 8.8 Hz, phenyl-C10H), 7.79 (1H, td, J = 7.7, 1.6 Hz, pyridyl-C3H),
8.13 (1H, s, triazole-H), 8.18 (1H, d, J = 7.9 Hz, pyridyl-C4H), 8.23 (2H, d,
J = 8.7 Hz, phenyl-C11H), 8.55 (1H, d, J = 4.3 Hz, pyridyl-C1H). 13C-NMR
(CDCl3, 125 MHz): δ 53.44 (C8), 120.45 (C4), 122.30 (C7), 123.29 (C2),
124.49 (C11), 128.89 (C10), 137.15 (C3), 141.58, 148.28 (C9/C12 could not
resolve), 149.42 (C6), 149.60 (C1), 149.97 (C5). HRMS m/z 282.0861
([M + H]+ requires 282.0991).
To a solution of L2a (232 mg, 516 μmol), in toluene (2 mL), was added [Re
(CO)5Br] (211 mg, 519 μmol). The solution was heated under reflux with
stirring for 30 min then allowed to stand and cool to 25°C. The yellow
liquid was decanted to remove an insoluble brown oil and concentrated
under reduced pressure. Residue was washed with pentane and dried in
vacuo to yield product as light yellow tacky solid (314 mg, 76%). 1H-NMR
(CDCl3, 500 MHz): δ 1.40 (9H, s, C1H3), 1.64 (2H, s,C5H2), 2.21–2.23 (2H, m,
C12H2), 3.13 (2H, d, J = 2.9 Hz, C4H2), 3.38–3.66 (12H, m, C6H2-C11H2), 4.64
(2H, qd, (J = 12.6, 6.3 Hz, C13H2), 4.85 (1H, s, NHBoc), 7.39 (1H, t,
J = 6.4 Hz, C19H), 7.92–7.98 (2H, m, C17H, C18H), 8.74 (1H, s, C14H), 8.97
(1H, d, J = 5.4 Hz, C20H). 13C-NMR (CDCl3, 125 MHz): δ 28.55 (C1), 29.50
(C12), 29.93 (C5), 38.40 (C4), 49.28 (C13), 66.39 (C11), 69.47 (C6), 70.26,
70.46 (C7-C10 could not resolve), 79.11 (C2), 122.21 (C17), 125.52 (C14),
125.68 (C19), 139.34 (C18), 148.54 (C15), 149.67 (C16), 153.32 (C20), 156.23
(C3), 188.74, 195.42, 197.03 (carbonyl C ≡ O) HRMS m/z 822.1125
([M + Na]+ requires 822.1124), m/z 800.1300 ([M + H]+ requires
800.1305), m/z 744.0675 ([M-tBu + H]+ requires 744.0679), m/z 720.2061
([M-Br]+ requires 720.2043), m/z 700.0779 ([M-Boc + H]+ requires
700.0780), m/z 664.1426 ([M-tBu-Br]+ requires 664.1417), m/z 620.1532
([M-Boc-Br]+ requires 620.1519).
2-(1-(4-isothiocyanato-benzyl)-1H-1,2,3-triazol-4-yl)pyridine (L3)
To a suspension of SnCl2 (700 mg, 3.69mmol) in dry ethanol (60mL)
sparged with N2 was added 5 (100 mg, 356 μmol), and the mixture was
heated under reflux for 18 h. The solution was allowed to cool to room
temperature, and the pH was adjusted to 8 with aqueous NaOH (10mL,
1 M). The mixture was then concentrated under reduced pressure, and
the white precipitate was removed by filtration and washed with dichlo-
romethane (4× 50mL). The combined organic washings were added to
the aqueous filtrate and separated. The aqueous layer was further
extracted with dichloromethane (2× 50 mL). The combined organic
extracts were washed with brine (50 mL), dried (Na2SO4) and concentrated
under reduced pressure followed by drying in vacuo to yield a brown waxy
oil (77mg, 86%), which was used without further purification.
To a solution of triethylamine (0.1mL) and the above product in
dichloromethane (20 mL) was added thiophosgene (0.1mL) at 0°C. The
red solution was stirred 0°C for 30min then aqueous NaOH (40 mL, 1M)
was added, and the biphasic mixture was warmed to room temperature
and stirred vigorously for 36 h. The organic layer was separated, and the
aqueous layer was further extracted with dichloromethane (50 mL). The
combined organic extracts were dried (MgSO4) and concentrated under
reduced pressure. The dark red oil was purified using silica gel
chromatography using a 1:1 mixture of EtOAc/petroleum spirits to yield a
light yellow solid of L3 (29 mg, 32%). 1H-NMR (CDCl3, 500 MHz): δ 5.63
(2H, s, triazole-CH2-phenyl), 7.19–7.22 (2H, m, phenyl-C11H), 7.25 (1H, ddd,
J = 7.5, 4.9, 1.2Hz, pyridyl-C2H), 7.33–7.35 (2H, m, phenyl-C10H), 7.75 (1H,
td, J = 7.7, 1.8Hz, pyridyl-C3H), 7.89 (1H, dt, J = 7.9, 1.1Hz, pyridyl-C4H),
8.18 (1H, s, triazole-H), 8.65 (1H, ddd, J = 4.9, 1.8, 1.0Hz, pyridyl-C1H).
13C-NMR (CDCl3, 125 MHz): δ 58.30 (C8), 120.86 (C4), 123.34 (C2), 126.24
(C11), 129.45 (C10), 131.58 (C9), 133.57 (C7), 134.42 (C12), 136.25 (C13),
136.96 (C3), 148.69 (C6), 149.78 (C5), 149.88 (C1). HRMS m/z 294.0810
([M+ H]+ requires 294.0813).
[Re(CO)3L2a(py)]OTf
To a solution of [Re(CO)3L2aBr] (64 mg, 80 μmol) in dichloromethane
(5 mL) was added AgOTf (21 mg, 82 μmol), and the suspension was
stirred for 2 h. The suspension was filtered through celite to remove
precipitated AgBr and then pyridine (20 μL, 248 μmol) was added, and
the solution was heated to reflux for 16 h. Solvent was removed in vacuo,
and the residue was washed with pentane to give product as slightly
yellow oil (74 mg, 97%). 1H-NMR (CDCl3, 500 MHz): δ 1.40 (9H, s, C1H3),
1.71 (2H, quintet, J = 6.3 Hz, C5H2), 2.33 (2H, dt, J = 13.0, 6.5 Hz, C12H2),
3.17 (2H, q, J = 6.2 Hz, C4H2), 3.49–3.64 (12, m, C6H2-C11H2), 4.74 (2H, t,
J = 7.1 Hz, C13H2), 5.00 (1H, s, NHBoc), 7.35 (2H, dd, J = 7.7 6.5 Hz,C23H),
7.62 (1H, ddd, J = 7.6, 5.7, 1.3 Hz, C20H), 7.83 (1H, tt, J = 7.7, 1.5 Hz, C24H),
8.15 (1H, td, J= 7.9, 1.4Hz, C19H), 8.22–8.24 (2H, m, C22H), 8.39 (1H, d,
J = 7.9Hz, C18H), 9.06 (1H, dd, J= 5.5, 0.6Hz, C21H) 9.37 (1H, s, C15H). 13C-
NMR (CDCl3, 125 MHz): δ 28.53 (C1), 29.75 (C5), 29.87 (C12), 38.57 (C4),
50.47 (C13), 67.21 (C11), 69.55 (C6), 70.25, 70.41, 70.52, 70.55 (C7-C10 could
not resolve), 78.98 (C2), 120.75 (q, JFC = 320.4Hz, CF3SO-3), 124.57 (C17),
1
127.04 (C22), 127.28 (C19), 127.98 (C14), 139.84 (C23), 141.73 (C18) 149.18
(C15), 149.88 (C16), 151.88 (C21), 152.63 (C20), 156.15 (C3), 190.60, 193.84,
195.91 (carbonyl C≡ O). HRMS m/z 799.2439 ([M]+ requires 799.2465). IR
(thin film) υ (CO) 2032, 1909 (broad, two peaks overlapped) cm-1.
Peptide conjugation
The cyclic-(RGDfK) peptide was synthesized according to literature
procedure.45 To a solution of cRGDfk (5.7 mg, 9.4 μmol) and DIPEA
(10 μL) in DMF (1 mL) was added L2b (8 mg) in two portions, and the
solution was stirred for 16 h at 25°C. The solvent was removed in vacuo,
and the crude residue was dissolved in acetonitrile/water (10%/90%)
(10 mL) and applied to a C18 semiprepartive HPLC column and separated
using the HPLC method previously outlined. Fractions containing pure
material were combined, frozen and lyophilized to yield desired product.
HRMS m/z 1145.5064 (C52H73N16O10S2 [M + H]+ requires 1145.5137),
573.2557 ([M + 2H]2+ requires 573.2608).
[Re(CO)3L1(py)]OTf
To a solution of MEOH (20 mL) was added [Re(CO)3(py)3]OTf (51 mg,
78 μmol) and L1 (19 mg, 80 μmol). The solution was heated under reflux
overnight with stirring. The yellow solution was concentrated under
reduced pressure, and the resulting precipitate was collected by filtration
and washed with diethyl ether. Crude material was recrystallised from
chloroform to yield product as yellow crystals (35 mg, 60%). 1H-NMR
(CDCl3, 500 MHz): δ 5.77 (2H, q, J = 10.7 Hz, C5H2), 7.23–7.25 (2H, m,
C14H), 7.40–7.47 (3H, m, C2H, C1H), 7.55–7.58 (3H, m, C3H, C11H), 7.80
(1H, tt, J = 7.7, 1.6 Hz, C15H), 8.11–8.16 (3H, m, C13H, C18H), 8.38 (1H, dt,
J = 8.0, 1.1 Hz), C9H), 9.02 (1H, ddd, J = 5.6, 1.5, 0.8 Hz, C12H), 9.45 (1H, s,
Ligand labelling with 99mTc
The preparation of [99mTc(CO)3(H2O)]+ was as follows: to an Isolink kitTM
vial (Mallinckrodt Medical B.V., The Netherlands) was added 1 mL of
99mTcO- eluted from a Gentech® generator (1000 MBq), and the vial
1
C6H). 13C-NMR (CDCl3, 125 MHz): δ 56.59 (C5), 120.62 (q, JFC = 320.2 Hz,
CF3SO3- ), 124.79 (C9), 126.95 (C14), 127.00 (C11), 127.79 (C6), 129.26 (C3), was heated to 100°C for 20 min. The contents of the vial was diluted by
129.56 (C2), 129.71 (C1), 133.06 (C4), 139.78 (C15), 141.68 (C10), 149.41 50 % and used for the subsequent labelling of ligands. The general
(C7), 150.09 (C8), 151.78 (C13), 152.32 (C12), 190.50, 193.79, 196.14 procedure for labelling is as follows: to a vial containing 0.1 mL of ligand
(carbonyl C ≡ O). HRMS m/z 586.1017 ([M]+ requires 586.0889), m/z solution (1 mg mL-1 ethanol) and 0.1 mL MilliQ water was added 0.1 mL of
507.0618 ([M-pyridine]+ requires 507.0467). IR (thin film) υ (CO) 2034,
[
99mTc(CO)3(H2O)]+. The mixture was then adjusted to pH 7.5 using 0.1 M
HCl, and the reaction mixture was heated to 75°C for 30 min. A solution of
1939, 1910 cm-1.
www.jlcr.org
Copyright © 2013 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2014, 57 262–269

T. U. Connell et al.
5% pyridine in MilliQ water (5 μL) was added, and the mixture was [19] S. Alves, J. D. G. Correia, L. Gano, T. L. Rold, A. Prasanphanich,
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heated to 75°C for a further 60 min.
X-ray crystallography. Crystals of the compound [Re(CO)3L1(py)]OTF
were mounted in low-temperature oil then flash cooled. Intensity data
were collected at 130 K on an X-ray diffractometer with CCD detector
using Cu-Kα (λ = 1.54184 Å). Data were reduced and corrected for
absorption.46 The structure was solved by direct methods and difference
Fourier synthesis using the SHELX suite of programs47 as implemented
within the WINGX48 software. Thermal ellipsoid plots were generated
using the program ORTEP-3.
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Crystal data for compound 5. C23H17N5O6 F3SRe M = 734.67, T = 130.1
(3) K, λ = 0.7107, Monoclinic, space group 21/n a = 10.9272(2),
b = 8.44830(10), c = 27.5153(5) Å, α = 90, β = 93.877(2), γ = 90(3), V= 2534.30
(7) Å3, Z = 4, Dc = 1.926 Mgm-3 μ(Cu-Kα) 4.949mm-1, F(000) = 1424, crystal
size 0.383 × 0.278 × 0.085 mm3. 37790 reflections measured, 10115
independent reflections (Rint = 0.0361), the final R was 0.0371 [I> 2σ(I)]
and wR(F2) (all data) was 0.0750. Cambridge Crystallographic Data Centre
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Acknowledgements
Funding from the Australian Research Council is acknowledged.
Ms Andrea North is thanked for assistance with technetium
chemistry and HPLC analysis; Mr Gordon Chan and Mr Kenneth
Young, (Centre for PET, Austin Hospital, Melbourne, Australia),
are acknowledged for help in providing 99mTc from the
generator. We also would like to acknowledge Covidien for
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providing samples of the Isolink KitTM
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Conflict of Interest
The authors did not report any conflict of interest.
[37] K. A. Stephenson, S. R. Banerjee, T. Besanger, O. O. Sogbein,
M. K. Levadala, N. McFarlane, J. A. Lemon, D. R. Boreham, K. P. Maresca,
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