Toward Hypoxia-Selective Rhenium and Technetium Tricarbonyl Complexes

Article abstract of DOI:10.1021/acs.inorgchem.5b01691

With the aim of preparing hypoxia-selective imaging and therapeutic agents, technetium(I) and rhenium(I) tricarbonyl complexes with pyridylhydrazone, dipyridylamine, and pyridylaminocarboxylate ligands containing nitrobenzyl or nitroimidazole functional g

Full text of DOI:10.1021/acs.inorgchem.5b01691

Article
pubs.acs.org/IC
Toward Hypoxia-Selective Rhenium and Technetium Tricarbonyl
Complexes
Andrea J. North,† David J. Hayne,† Christine Schieber,† Katherine Price,‡ Anthony R. White,‡
Peter J. Crouch,‡ Angela Rigopoulos,⊥,#,∇ Graeme J. O’Keefe,§ Henri Tochon-Danguy,§
Andrew M. Scott,§,∥,⊥,# Jonathan M. White,† Uwe Ackermann,§,∥,⊥,# and Paul S. Donnelly*,†
†School of Chemistry and Bio21 Molecular Science & Biotechnology Institute, ‡Department of Pathology, University of Melbourne,
Melbourne, Victoria 3010, Australia
∥
§Department of Molecular Imaging and Therapy, Department of Medicine, University of Melbourne, Austin Health, Studley Rd,
Heidelberg, Victoria 3010, Australia
#
⊥Olivia Newton-John Cancer Research Institute, School of Cancer Medicine, La Trobe University, Melbourne, Victoria 2086,
Australia
∇Ludwig Institute for Cancer Research, Melbourne-Austin Branch, 145 Studley Road, Heidelberg, Victoria 3084, Australia
S
* Supporting Information
ABSTRACT: With the aim of preparing hypoxia-selective
imaging and therapeutic agents, technetium(I) and rhenium(I)
tricarbonyl complexes with pyridylhydrazone, dipyridylamine,
and pyridylaminocarboxylate ligands containing nitrobenzyl or
nitroimidazole functional groups have been prepared. The
rhenium tricarbonyl complexes were synthesized with short
reaction times using microwave irradiation. Rhenium tricar-
bonyl complexes with deprotonated p-nitrophenyl pyridylhy-
drazone ligands are luminescent, and this has been used to
track their uptake in HeLa cells using confocal fluorescent
microscopy. Selected rhenium tricarbonyl complexes displayed
higher uptake in hypoxic cells when compared to normoxic
cells. A 99mTc tricarbonyl complex with a dipyridylamine ligand
bearing a nitroimidazole functional group is stable in human serum and was shown to localize in a human renal cell carcinoma
(RCC; SK-RC-52) tumor in a mouse.
hydroxylamine with reactive biomolecules found inside cells. In
normal cells, it is argued that any reduced intermediates are
rapidly oxidized back to the original nitroimidazole, which is
then capable of diffusing back out of the cell. Although FMISO
has demonstrated considerable potential as a hypoxia imaging
agent, it suffers from relatively slow cell uptake and poor
clearance from normoxic tissue.4−6
While the number of hospitals equipped with PET
infrastructure is increasing, single photon emission computed
tomography (SPECT) remains the major technique for nuclear
medicine imaging. The most commonly used radioisotope for
SPECT is technetium-99m (99mTc), and unlike 18F that
requires a cyclotron for production of tracers, 99mTc is readily
available from commercial generators. The nuclear properties of
99mTc (t1/2 = 6.01 h, Eγ = 141 keV) are ideally suited to
diagnostic procedures. A hypoxia-selective 99mTc radiopharma-
ceutical that utilizes the more commonly encountered imaging
modality of SPECT could be an attractive alternative to 18F
INTRODUCTION
■
Tissue hypoxia is a complicated condition in which O2 demand
exceeds supply and results in disrupted cellular metabolism. It
can occur when vascular dioxygen supply is interrupted after
stroke or myocardial infarction, or when a tumor exceeds the
vascular diffusion limit for oxygen. Tumors that are hypoxic can
be more resistant to traditional therapeutic strategies. Imaging
agents that are selectively retained in hypoxic tissue can be
detected using noninvasive molecular imaging techniques and
have a role to play in increasing the understanding of hypoxia in
tumor biology. Hypoxia imaging agents are also useful tools for
clinicians when designing and assessing treatment regimes.1
The molecular imaging technique of positron emission
tomography (PET) relies on a positron-emitting molecule, a
tracer that is administered to a patient, and the emitted
radiation is detected by an external camera. A 2-nitroimidazole,
labeled with positron-emitting fluorine-18, 18F-fluoromisonida-
zole (FMISO; Figure 1a), has been studied as a hypoxia
imaging agent.1−3 It is thought that FMISO is trapped
preferentially in hypoxic tissue by virtue of reduction of the
nitro functional group, followed by reaction of the reduced
Received: July 26, 2015
© XXXX American Chemical Society
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Figure 1. Chemical structures of (a) 18F-FMISO, (b) [TcO(PnAO-1-(2-nitroimidazole)] (BMS 181321), (c) [TcO(BnAO)] (Prognox), and (d)
hypoxia-selective nitrobenzyl prodrug, N,N-bis(2-chloroethyl)-N-methyl-N-(2-nitrobenzyl)ammonium chloride.
based 2-nitroimidazole systems. Efforts to produce hypoxia-
selective technetium complexes have focused on propylene
amine oxime ligands (PnAO) with appended nitroimidazole
functional groups such as [TcO(PnAO-1-(2-nitroimidazole)]
(Figure 1b). This complex selectively accumulates in hypoxic
cells in vitro; however its use in vivo is limited by high blood
uptake and extensive hepatobiliary excretion.7−9 The {TcVO}3+
complex of a butylene amine oxime ligand, [TcO(BnAO)]
(also referred to as [TcO(HL91)] or Prognox; Figure 1c), does
not contain a nitro functional group but does display some
degree of hypoxia selectivity, although the origins of this
specificity remain uncertain.10−12 Peptide-based ligands con-
taining a 2-nitroimidazole functional group for the {TcVO}3+
fragment have also been investigated.13,14
Bioreductive prodrugs that can undergo enzymatic reduction
are generally classified within one of five categories: the
nitroaromatics, quinones, aromatic/aliphatic N-oxides, or metal
complexes.15 Similarly to 2-nitroimidazoles, nitrobenzene-
containing agents are contained within the nitroaromatic
subset. These molecules are under continuing investigation
for their involvement in enzyme-catalyzed prodrug therapy,
where endogenous enzymes activate (reduce) the nitro
functional group to stimulate the formation of a cytotoxic,
bioactive molecule. This is demonstrated by the hypoxia-
selective antitumor arylmethyl quaternary mustard, N,N-bis(2-
chloroethyl)-N-methyl-N-(2-nitrobenzyl)ammonium chloride
(Figure 1d), which undergoes reductively induced fragmenta-
tion reactions via a radical intermediate.16,17 Nitrobenzyl-
containing rhenium and technetium complexes may be equally
as effective as 2-nitroimidazole-based complexes.
In this paper, we describe the synthesis and characterization
of fac-{MI(CO3)}+ (M = Re, 99mTc) complexes with aromatic
amine bi- and tridentate ligands containing nitroaromatic
functional groups with a view to producing hypoxia-selective
agents. Aromatic amines coordinate to the metal ion with rapid
complexation kinetics, and the resulting complexes possess high
stability.18 Though much emphasis has focused on the stability
of tridentate chelators, the [2 + 1] mixed-ligand option may
also be a viable approach due to the strong-field nature of the
fac-{MI(CO)3}+ (M = Tc, Re) core. A potential advantage of [2
+ 1] mixed-ligand complexation is that the apical [+1] site can
be changed to modulate the physical characteristics of the
compound such as the solubility, lipophilicity, and ultimately
membrane permeability.
The use of a single tridentate ligand has potential benefits in
the ease of radiochemical synthesis as well as enhanced complex
stability. Dipicolylamine,20−23 picolylamine monoacetic
acid,23−25 and iminodiacetic acid23,26,27 are commonly used
tridentate ligands to form complexes with fac-{MI(CO)3}+ (M
= Tc, Re), and a series of iminodiacetic acid-based ligands
featuring 2-nitroimidazole functional groups have been
investigated as potential hypoxia tracers.26−29
In this study, a series of rhenium complexes containing either
nitroaromatic or 2-nitroimidazole functional groups are
prepared and characterized. The cell uptake of the rhenium
compounds in both normoxia and under hypoxic conditions
was investigated using inductively coupled plasma mass
spectrometry (ICP-MS) and confocal fluorescence microscopy.
The technetium-99m complexes have been prepared and
characterized by comparison to the nonradioactive rhenium
analogues and one example has been assessed in a hypoxic
tumor model.
An alternative to the {TcVO}3+ core is the fac-{TcI(CO)3}+
precursor. Seminal developments in synthetic methods that
allow the preparation of technetium compounds of the type fac-
[Tc(CO)3(H2O)3]+ under conditions amenable to radio-
pharmaceutical applications have stimulated the focus on this
approach and reinvigorated research into targeted technetium
radiopharmaceuticals.18 The “tricarbonyl core” approach
exploits the stability of the metal tricarbonyl core while
manipulating the relatively labile water ligands to attach
targeting vectors. A variety of mono-, bi-, and tridentate ligands
can react with the fac-{TcI(CO)3}+ fragment and displace the
substitutionally labile aquo ligands.
RESULTS AND DISCUSSION
■
Synthesis and Characterization of [2 + 1] Mixed-
Ligand Ligands and Complexes. Two potentially bidentate,
2-pyridylhydrazone ligands, HL1 and HL2 (Figure 2), each
bearing a nitro functional group, were prepared by
condensation of 2-hydrazinopyridine with either p-nitro-
benzaldehyde or m-nitrobenzaldehyde. The ligands were
characterized by NMR spectroscopy, ESI-MS, and X-ray
crystallography.
The lack of nonradioactive isotopes of technetium
complicates exploratory chemical synthesis. Consequently,
rhenium, the nonradioactive, group VII congener of techne-
tium, is often used as a 99mTc surrogate in developmental
chemistry. Rhenium and technetium have similar ionic radii,
and {MI(CO)3}+ (M = Tc, Re) complexes are often
isostructural. The interest in rhenium analogues also extends
to the possibility of using the β−-emitting rhenium isotopes
186Re (t1/2 = 90.6 h, Emax = 1.1 MeV, 91%) and 188Re (t1/2
=
Figure 2. Bidentate ligands, HL1−HL4, and tridentate ligands, L5 and
HL6.
17.0 h, Emax = 2.1 MeV, 85%) for radiotherapeutic
applications.19
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X-ray crystallography of HL1 and HL2 (Figure 3) reveals that
both compounds crystallize in the E conformation. H NMR
essentially quantitative yields and significantly reduced reaction
times (Scheme 1).
1
Complexes fac-[Re(CO)3(HL1)Br] and fac-[Re(CO)3(HL2)-
Br] were synthesized by the addition of equimolar amounts of
the ligands to a mixture of fac-[Re(CO)3(OH2)3]Br in
methanol (Scheme 1). The major signal in the high resolution
ESI-MS of both complexes, with the predicted isotope pattern,
was for the cation formed by a loss of coordinated bromide at
m/z = 513.0203 (calculated 513.0203). The resonances
attributed to the hydrazinic NH proton in the 1H NMR
spectra of the complexes fac-[Re(CO)3(HL1)Br] and fac-
[Re(CO)3(HL2)Br] are shifted downfield when compared to
the “free” ligands, for example, NH(hydrazinic) δ 13.2 ppm for
fac-[Re(CO)3(HL1)Br] compared to δ 9.84 ppm for HL1. The
structures of fac-[Re(CO)3(HL1)Br] and fac-[Re(CO)3(HL2)-
Br] were confirmed by X-ray crystallography (Figure 4).
Replacing the monodentate [+1] bromide ligand can lead to
the modification of physical and biological properties of the
metal and also produce complexes that are more resistant to
substitution in vivo. It was of interest to use pyridine based
ancillary [+1] ligands due to the ability of heterocyclic amines
to form complexes that are resistant to substitution, and the use
of 3-(dimethylamino)-1-propylamine (DMAPA) as an ancillary
ligand was designed to dramatically change solubility and
membrane permeability.
Figure 3. ORTEP representations of nitro-bearing ligands (a) HL1
and (b) HL2 with thermal ellipsoids shown at 50% probability.
Hydrogen atoms have been omitted unless relevant.
spectroscopy suggests that this conformation is retained when
HL1 and HL2 are dissolved in d6-DMSO, as in both cases the
hydrazinic proton atom gives a broad resonance at δ 10.4−11.3
ppm.
The [NEt4]2[Re(CO)3Br3] precursor is commonly used to
prepare complexes featuring a fac-{ReI(CO)3}+ core and is
often synthesized by reacting [Re(CO)5Br] with excess
tetraethylammonium bromide in diglyme at 180 °C. An
alternative starting material, fac-[Re(CO)3(H2O)3]+, can be
prepared by heating [Re(CO)5Br] at reflux in water for 24 h.
This more convenient method is moderately successful, but the
relative insolubility of the [Re(CO)5Br] starting material in
water and its hydrophobicity leads to a substantial loss in yield
when working on smaller scales (ca. < 100 mg) as the rhenium
pentacarbonyl bromide tends to coat glassware and require
constant “washing” back into the reaction mixture. During the
course of this work, we have developed a microwave heating
method of producing fac-[Re(H2O)3(CO)3]+ in near quantita-
tive yields that is equally effective on small scales as on larger
scales. Commercially available [Re(CO)5Br] was suspended in
water and the mixture sparged with nitrogen and subjected to
microwave-assisted thermal heating for three intervals of 10
min to give clear solutions of fac-[Re(CO)3(OH2)3]Br in
Initial synthesis used a high-boiling solvent, 1,2-dichlor-
obenzene, and an excess of ligand. A superior synthetic
procedure involved the reaction of equimolar amounts of
ligand, HL1 or HL2, and tris-substituted fac-[Re(CO)3(X)3]+ (X
= pyridine, 4-picoline, 3-(dimethylamino)-1-propylamine) in
methanol via microwave irradiation for 20 min (Scheme 1).
Crystals of fac-[Re(CO)3(L1−2)(X)], suitable for X-ray analysis,
were formed following partial evaporation of the reaction
mixture solvent, in yields of approximately 70% (Figure 5,
Table 4). Replacing the monodentate bromide ligand with basic
N-containing heterocycles, pyridine or 4-picoline, resulted in
the isolation of neutral complexes with the deprotonation of the
bidentate hydrazone ligand.
It is interesting to note that fac-[Re(CO)3(HL1)(pyridine)]-
BPh4 is the only complex to crystallize as the cationic species
via protonation of the hydrazinic (N2) nitrogen (Figure 5a);
nonetheless deprotonation is easily induced by the addition of
Scheme 1. Reaction Schemes for the Synthesis of fac-[Re(CO)3(OH2)3]Br, fac-[Re(CO)3(HL1−4)Br], and fac-
[Re(CO)3(L1−2)(X)] (X = pyridine, 4-picoline, DMAPA) Complexes
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N1−Re torsion angles, whereby the cationic complex fac-
[Re(CO)3(HL1)(pyridine)]+ possesses an angle of 19.0(8)°,
and torsion angles for the three complexes deprotonated at the
N2 nitrogen (fac-[Re(CO)3(L2)(pyridine)] and fac-[Re-
(CO)3(L1,2)(4-picoline)]) lie between 174.4 and 179.0°
(Figure 5, Table 1).
The Re−N4 bond lengths are predictably shorter than the
Re−Br distances for the fac-[Re(CO)3(HL1−2)(Br)] series of
complexes, with an average distance of 2.626 Å for the two
nitro-substituted fac-[Re(CO)3(HL1−2)(Br)] complexes and
2.212 Å for the N-heterocycle substituted complexes (Table 1).
Additionally, the Re−N1 distance is also consistently shorter
for complexes with nitroaromatic coordination at the ancillary
site, compared to the bromo-substituted rhenium complexes
(2.22 vs 2.16 Å).
The N1−C7 sp2 hybridized bond for the two nitro-bearing
ligands (HL1−2) in (fac-[Re(CO)3(L1−2)Br]) and fac-[Re-
(CO)3(L1−2)(X)] (X = pyridine, 4-picoline) does not vary
significantly in length. The N1−C7 distances for ligands HL1
and HL2 are 1.278(2) and 1.279(3) Å, respectively. A slight
increase in this bond length is observed upon coordination as
the apical, monodentate ligand is varied from bromo, pyridine,
or picoline coordination. For all neutral, [2 + 1] complexes
without a coordinated bromide, the Re−N1 bonds of the five-
membered chelate ring are shorter (2.16 vs 2.22 Å), and Re−
N3 and C8−N3 bond lengths are longer when compared to the
analogous complexes with a coordinated anionic bromide (1.37
vs 1.34 Å). N1−N2 bond lengths of fac-[Re(CO)3(HL1)(Br)]
and fac-[Re(CO)3(L1)(4-picoline)] (average 1.356 Å) tend
toward the distance of the N1−N2 ligand bond (average 1.358
Figure 4. ORTEP representations of complexes (a) fac-[Re-
(CO)3(HL1)Br] and (b) fac-[Re(CO)3(HL2)Br], with thermal
ellipsoids shown at 50% probability. Hydrogen atoms have been
omitted unless bound to nitrogen.
triethylamine. The differences between the protonated and
nonprotonated N2 derivatives are exemplified in the C1−C7−
Figure 5. ORTEP representations of (a) fac-[Re(CO)3(HL1)(pyridine)]BPh4, (b) fac-[Re(CO)3(L2)(pyridine)], (c) fac-[Re(CO)3(L1)(4-
picoline)], (d) fac-[Re(CO)3(L2)(4-picoline)] with thermal ellipsoids shown at 50% probability. Hydrogen atoms, counterion, and solvent
molecules have been omitted for clarity unless relevant.
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The resonance of the hydrazinic NH signals in the proton
NMR of the ligands and fac-[Re(CO)3(HL1−4)(Br)] complexes
demonstrate a shift upfield once the ligand coordinates the
metal; for instance, the “free” ligand HL1 displays an NH
resonance at δ 9.84 ppm which shifts to δ 13.2 ppm for the
complex fac-[Re(CO)3(HL1)Br]. Hydrazinic proton shifts are
not evident for the picoline, pyridine, or DMAPA complexes,
and the absence of the hydrazinic υ(N−H) infrared stretch,
Table 1. Selected Bond Lengths (Å) and Angles (deg) for
(1) fac-[Re(CO)3(HL1)(pyridine)]BPh4 (2) fac-
[Re(CO)3(L2)(pyridine)] (3) fac-[Re(CO)3(L1)(4-
a
picoline)] (4) fac-[Re(CO)3(L2)(4-picoline)]
1
2
3
4
bond lengths
Re−N(4)
Re−N(1)
2.201(4)
2.165(4)
2.195(4)
1.932(6)
1.937(5)
1.909(6)
1.379(6)
1.362(7)
1.361(6)
1.294(6)
2.217(5)
2.161(4)
2.134(4)
1.935(5)
1.905(5)
1.921(6)
1.380(5)
1.329(6)
1.374(7)
1.295(6)
2.216(3)
2.159(3)
2.134(3)
1.919(4)
1.932(3)
1.931(4)
1.354(4)
1.351(5)
1.365(5)
1.319(5)
2.215(4)
2.155(4)
2.147(4)
1.936(5)
1.912(4)
1.923(5)
1.379(5)
1.342(6)
1.376(6)
1.319(6)
which is apparent in the protonated complexes between ν 3182
̅
Re−N(3)
and 2931 cm−1, provides support that these complexes are
deprotonated.
Re−C(13)
Re−C(14)
Re−C(15)
N(1)−N(2)
N(2)−C(8)
N(3)−C(8)
N(1)−C(7)
bond angles
Infrared CH stretches for the monodentate pyridine ring
and the azomethine are shifted to a slightly higher energy
compared to the “free” ligand, which is consistent with the
decrease in electron density within the rings and backbone,
though overlap of the absorbances from the pyridine skeletal
bands and υ(NO2) stretches cause some obscurity.
The infrared spectra for fac-[Re(CO)3(H2O)3]+ and the tris-
picoline and tris-pyridine complexes consistently display two
typical stretching bands for the facially orientated terminal
N(1)−Re−
75.15(16)
99.79(18)
90.6(2)
74.08(15)
98.97(19)
87.6(2)
73.89(12)
97.47(14)
88.09(15)
98.57(13)
171.42(13)
171.20(13)
177.44(13)
92.30(14)
92.42(14)
85.03(11)
82.95(11)
90.12(15)
88.09(15)
94.21(14)
94.49(13)
74.25(14)
98.07(17)
86.42(19)
96.45(17)
169.95(17)
171.72(17)
177.53(16)
91.33(17)
92.00(17)
84.81(13)
83.50(13)
87.0(2)
N(3)
N(3)−Re−
tricarbonyl, υ(CO), moiety at ν 2040 and 1881 cm−1. All
C(14)
̅
C(14)−Re−
other complexes which deviate from C3v symmetry show either
C(13)
two or three bands between ν 2016 and 1894 cm−1 in the
̅
C(13)−Re−
100.1(2)
174.6(2)
172.67(18)
174.65(19)
94.00(18)
93.78(19)
80.20(16)
82.80(15)
89.3(2)
98.55(18)
172.18(18)
172.83(19)
176.4(2)
93.86(19)
93.57(19)
83.70(15)
83.15(15)
89.7(2)
typical carbonyl stretching region of compounds containing α-
diimine ligands.30 It is assumed that those complexes which
display only two stretches have the middle band obscured by
the stronger, broad stretch at the lower energy.
N(1)
C(13)−Re−
N(3)
C(14)−Re−
N(1)
Electrochemical Studies. The electrochemical character-
istics of the nitro-containing ligands (HL1 and HL2) and all [2
+ 1] mixed-ligand complexes were studied by cyclic
voltammetry in acetonitrile. Potentials are quoted relative to
the ferrocene/ferrocenium couple, where E0′ is designated as
+0.40 V vs SCE.31 The cathodic region was investigated for all
compounds in order to establish the location of significant
redox processes. Comparative redox potentials were examined
against the para-methoxy substituted complex fac-[Re-
(CO)3(L3)Br] and unsubstituted phenyl derivative fac-[Re-
(CO)3(L4)Br] (where R1 = −OCH3 or −H).
C(15)−Re−
N(4)
C(14)−Re−
N(4)
C(13)−Re−
N(4)
N(1)−Re−
N(4)
N(3)−Re−
N(4)
C(13)−Re−
C(15)
C(14)−Re−
90.6(2)
87.6(2)
86.42(19)
97.56(16)
97.81(16)
For nitro-containing complexes fac-[Re(CO)3(HL1)Br] and
fac-[Re(CO)3(HL2)Br], two reduction waves are observed
within the range 0.0 and −1.15 V. The first peak is generally
irreversible and the second quasi-reversible; this is a common
observation for nitro-aromatic compounds,32,33 although this
pattern was not seen for either of the nitro-based ligands which
display only one quasi-reversible peak.
C(15)
C(15)−Re−
94.95(19)
93.81(18)
94.45(19)
93.4(2)
N(1)
C(15)−Re−
N(3)
a
Complexes are listed in order of appearance from Figure 5.
Complexes containing the nitro substituent in the para
position of the ligand (L1) consistently display higher reduction
potentials and more complex voltammograms than the meta-
nitro (L2) species. The broadness of the first reduction wave for
fac-[Re(CO)3(L1−2)Br] and fac-[Re(CO)3(L2)(X)] (X =
pyridine, 4-picoline) indicates that multiple electron processes
are occurring. This is further confirmed by the occurrence of
three distinct reduction peaks for fac-[Re(CO)3(L1)(pyridine)]
between −0.80 and −1.18 V (Table 2).
It is worth noting that complexes with nitrogen substituted
[+1] ancillary ligands are deprotonated at the hydrazinic
nitrogen in solution, if not confirmed by the crystal structure
data but also by the change in color to the analyte solution to
deep red. This indicates that the first cathodic peak most likely
involves a proton-coupled reduction, which, though ligand
based, is affected by coordination to the metal to give an
electrochemical−chemical based process.
Å), while all other complexes consistently show slightly longer
lengths (between 1.379 and 1.382 Å).
The 1H NMR spectra of the ligand and complexes with an m-
nitro substituent (HL2, fac-[Re(CO)3(HL2)(Br)], fac-[Re-
(CO)3(L2)(4-picoline)], fac-[Re(CO)3(L2)(pyridine)], and
fac-[Re(CO)3(L2)(DMAPA)]) were all as expected. For
example, coordination of the metal ion to HL2 results in a
significant downfield shift in the resonance attributed to the
proton attached to C2 (Figure 4b), shifting from δ 8.47 ppm
for HL2 to δ 9.66 ppm for the complex fac-[Re(CO)3(L2)(4-
picoline)]. Additionally, for all picoline, pyridine, and DMAPA
complexes, a dramatic shift to a higher field for the resonance
attributed to the CHN proton is observed. As this shift is not
reflected in the bromo complexes, it can be assumed that the
shielding effects of the nitrogen-containing moiety are
substantial.
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Table 2. Electrochemical Data for Selected Ligands and
a
Complexes
i
/
ΔE
p e
b
c
pa(qr)d
Epc1 (V)
Epc(qr) (V)
ipc(qr)
(mV)
HL1
HL2
−1.10
−1.18
−1.05
3.38
3.00
0.87
58
58
62
fac-[Re(CO)3(HL1)
−0.65
−0.74
−1.50
−1.32
Br]
fac-[Re(CO)3(HL2)
−1.14
1.38
64
Br]
fac-[Re(CO)3(HL3)
Br]
fac-[Re(CO)3(HL4)
Br]
fac-[Re(CO)3(L1)(4-
−0.98
−0.99
0.74
0.90
1.29
0.87
60
64
63
56
pic)]
Figure 6. Cyclic voltammograms of selected meta-NO2 compounds.
Experimental conditions: 1.0 mM analyte in 100 mM [tBu4N][BF4]/
CH3CN electrolyte solution, scan rate 100 mV s−1.
fac-[Re(CO)3(L1)
−0.80
−0.71
f
(py)]
(−1.18)
−1.14
fac-[Re(CO)3(L2)(4-
f
pic)]
(−0.85)
−0.81
(CO)3(L5)]BPh4 and fac-[Re(CO)3(L6)] took advantage of
the previously reported synthesis of 2-nitroimidazole by initial
diazatization from 2-aminoimidazole sulfate, and subsequent
nitration was performed using copper sulfate as a catalyst.36
Bromoalkylation of the potassium salt of the nitroimidazole was
obtained according to published procedure using 18-crown-6 in
anhydrous acetonitrile to afford the desired bromobutylimida-
zole (1).37
fac-[Re(CO)3(L2)
−1.13
(py)]
a
Potential measurements of all complexes are referred to the SCE,
with 1.0 mM analyte in CH3CN/100 mM [Bu4N][BF4] relative to the
Fe0/Fe1 redox couple, E0′ = +0.40 V (vs SCE) at a 100 mV s−1 scan
b
rate Cathodic peak potential corresponding to the first reduction
step. Where there is no value given, the first cathodic peak is quasi-
reversible, and values are provided in the proceeding column.
c
Formation of the tridentate ligand, L5, involved a substitution
reaction of bromoalkylated nitroimidazole 1 with di(2-picolyl)-
amine and N,N-diisopropylethylamine (DIPEA) as a base. A
solution of ligand L5 and [Re(CO)5OTf] in methanol was
subjected to microwave irradiation at 120 °C for 15 min and
precipitated with sodium tetraphenylborate in methanol,
resulting in the formation of the desired complex fac-
[Re(CO)3(L5)]BPh4. The complex was crystallized by
evaporation of a solution of the complex in methanol, to
afford clear, block crystals in a moderate yield (56%; Scheme
2).
Cathodic peak potential corresponding to the second quasi-reversible
d
(qr) reduction step. Peak current ratio of second quasi-reversible
cathodic peak. ΔEp = |Epc − Epa| of quasi-reversible peak (voltage
separation between current peaks). Potentials in brackets indicate a
visibly distinct reduction potential between or after indicated reduction
peaks. Abbreviations: pc = cathodic potential, pa = anodic potential, qr
= quasi reversible.
e
f
The latter, quasi-reversible cathodic peak is assigned to an
initial one-electron reduction of the nitro conjugate base to the
radical anion.5,7,32,34 All quasi-reversible reduction processes
show a peak separation (ΔEp) value between 58 and 64 mV
indicating a one-electron reversible process. Peak current ratios
(ip/ia) are more variable, with ratios for the complex quasi-
reversible reduction processes between 0.74 and 1.38. All quasi-
reversible reduction processes for the rhenium complexes were
measured at scan rates of 50, 100, 200, and 300 mV s−1 and
produce linear correlations (R2 ≈ 0.99) when plotted against
the square root of the scan rate (v1/2).
A similar procedure for the formation of the neutral complex
fac-[Re(CO)3(L6)] was used; however synthesis of the ligand,
HL6, involved the initial preparation of 2 via condensation of 2-
picolylamine and ethylbromoacetate followed by Kugelrohr
distillation and reaction with the bromoalkylated nitro-
imidazole, 1. In situ hydrolysis of the ester using sodium
hydroxide and thermal heating at 120 °C with [Re(CO)5OTf]
in acetonitrile afforded the desired complex fac-[Re(CO)3(L6)],
in good yield as an off-white solid (76%).
The redox chemistry for the meta-nitro ligand (HL2)
compared to corresponding rhenium complexes displays a
slight shift to higher potential values upon coordination,
whereby the uncomplexed ligand shows an Epc value of −1.18 V
and the complexes have reduction potentials of −1.14 V
(Figure 6); this is tentatively assigned to the slightly electron-
withdrawing nature of the metal center.7
For similar rhenium compounds, the metal-centered
reduction assigned to Re1/Re0 is between −1.2 and −1.6 V
and devoid of an oxidation wave (CH3CN, 100 mM
tetrabutylammonium perchlorate).35 It is most likely that,
within the observed window, experimental potentials are not
inclusive of the reduction of rhenium. Furthermore, the
reduction of rhenium(I) to rhenium(0) is not likely to occur
in the cellular environment.
High resolution ESI mass spectrometry of both ligands, L5
and HL6, indicates consistency with all spectroscopic data; for
instance, the pure ligand, L5, was obtained with an m/z peak at
353.1723, assigned to [L5+H]+ (calculated 353.1726). Both
complexes clearly show the rhenium isotopic pattern at their
expected m/z peaks.
IR spectra of both tridentate complexes demonstrate
stretching bands between ν 2027 and ν 1874 cm−1, indicating
̅
̅
the presence of the fac-{Re(CO)3}+ core. The carbonyl stretch
for the esterified ligand, L6Et (ν 1730 cm−1), is within the
̅
typical range,38 but upon complexation a reduced frequency of
absorption is observed for the neutral complex fac-[Re-
(CO)3(L6)], with absorptions at ν 1652 and 1365 cm−1.
̅
These two bands are believed to denote carboxylate asym-
...
metrical and symmetrical stretching bands, υas(C − O)2− and
...
Synthesis and Characterization of Tridentate Ligands
and Complexes. The preparation of the ligands involved in
the formation of the two tridentate complexes fac-[Re-
υs(C − O)2− respectively, and are not seen for the cationic
structure fac-[Re(CO)3(L5)]+.
F
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Scheme 2. Synthesis of Tridentate Complexes fac-[Re(CO)3(L5)]+ and fac-[Re(CO)3(L6)]
Coordination of the fac-{Re(CO)3}+ core to L5 results in a
significant downfield shift in resonances in both the H and
Table 3. Selected Bond Lengths (Å) and Angles (deg) for the
1
a
Rhenium Complex fac-[Re(CO)3(L5)]+
13C{1H} NMR spectra, particularly for the equivalent
methylene protons adjacent to the pyridine rings which shift
from a singlet resonance at δ 3.74 ppm for the ligand to δ 4.60
ppm for the complex; this is most likely due to the deshielding
effects of the rhenium center. Similar patterns are observed for
the HL6 ligand and complex. Evidence advocating the
formation of the neutral complex shows transformation of the
methylene protons adjacent to the pyridine and carboxylate
from two singlet resonances to a distinctive set of four doublets
indicating geminal coupling (2JHH = 16 Hz); this does not occur
for the cationic complex.
Bond Lengths
Re−
C(19)
1.927(3) Re−N(1) 2.184(3) C(19)−
1.147(4)
1.152(4)
1.151(4)
O(3)
Re−
C(20)
1.913(3) Re−N(2) 2.165(2) C(20)−
(O5)
Re−
C(21)
1.922(4) Re−N(3) 2.227(2) C(21)−
(O4)
Bond Angles
N(1)−Re−C(21)
N(2)−Re−C(19)
N(3)−Re−C(20)
C(19)−Re−N(1)
C(19)−Re−N(3)
C(19)−Re−C(21)
173.63(10)
174.03(12)
173.52(10)
96.18(12)
98.33(11)
85.48(14)
N(1)−Re−N(3)
N(1)−Re−N(2)
N(3)−Re−N(2)
C(20)−Re−N(1)
C(20)−Re−N(2)
C(20)−Re−C(21)
77.34(9)
78.30(9)
78.36(9)
99.03(12)
95.70(110
87.18(13)
Crystals suitable for analysis by X-ray crystallography for fac-
[Re(CO)3(L5)]BPh4 were grown from a concentrated solution
of the compound in methanol (Figure 7). The complex
a
Bond distances and angles for the Ph4B counterion have not been
shown.
both nitro ligand species, L5 and L6Et, respectively. The quasi-
reversible reduction wave for the complexes is shifted to −1.08
V for the cationic fac-[Re(CO)3(L5)]+ complex and −1.12 V
for fac-[Re(CO)3(L6)]. No initial, nonreversible reduction peak
was observed for the complexes, suggesting that any other
reduction processes occur outside of the observed window.
UV−Visible and Fluorescence Studies. The absorption
spectra of both HL1 and HL2 dissolved in acetonitrile display
two peaks in the UV region, HL1 at λ 387 and 254 nm and HL2
at λ 327 and 225 nm, and are assigned to π → π* transitions. In
the metal complexes featuring a meta-nitro functional group,
MLCT transitions at λ 463−469 nm are evident that shift to λ
520−528 nm for complexes possessing the para-NO2 func-
tional group. These values reflect those of similar low-valent,
rhenium complexes, albeit more often as the α-diimine rather
than the hydrazone functionality.40 The low energy absorbance
demonstrates the strong π acceptor nature of the ligands, and
the bathochromic shift of the para-NO2 complexes implies the
delocalization of electron density caused by the para
substituent.
Figure 7. ORTEP representation of fac-[Re(CO)3(L5)]+ with thermal
ellipsoids shown at 50% probability. Hydrogen atoms, counterion, and
solvent molecules have been omitted for clarity.
crystallizes in the triclinic space group, P1, and displays
̅
characteristic crystallographic parameters, bond lengths and
angles for a tricarbonyl, tridentate rhenium complex (Table 3,
Table 4).25,26,39
Cyclic voltammetry shows a reduction peak pattern similar to
those seen for the [2 + 1] mixed-ligand complexes. A quasi-
reversible cathodic peak was observed at −1.10 and −1.11 V for
Complexes where the pyridyl-hydrazone is deprotonated and
contains the para-NO2 substituent fac-[Re(CO)3(L1)(X)] (X =
G
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H
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4-picoline, pyridine, DMAPA) are luminescent, whereas meta-
nitro species are not. The red-purple, para-nitro-substituted
complex fac-[Re(CO)3(L1)(pyridine)] absorbs at a wavelength
of 520 nm, with an emission at λ 715 nm (Figure 8). These
line with the aid of fluorescence microscopy. The cells were
exposed to increasing concentrations of the compound (5−50
μM) for 30 min, and fluorescence was measured following
excitation at a wavelength of 520 nm. The complex localizes
primarily within the cytosol with no uptake in the nucleus. The
complex also accumulated in puncta indicative of cytosolic
vesicles, and the cells retained their morphology during the 30
min incubation (Figure 9).
The cellular permeability and retention of fac-[Re-
(CO)3(HL1)Br], fac-[Re(CO)3(L1)(4-picoline)], fac-[Re-
(CO)3(L1)(DMAPA)], and fac-[Re(CO)3(L5)]BPh4 was as-
sessed in human neuroblastoma SH-(SY5Y) cells. The cells
were grown to 90% confluency, then incubated at 37 °C for 1 h,
with a total rhenium complex concentration of 10 μM
maximum. The intracellular rhenium concentrations were
determined using ICP-MS. Basal rhenium levels were negligible
in the DMSO vehicle control and 0.06 μM for the standard,
rhenium-triflate complex [Re(CO)5(CF3SO3)]. Significant
increases in intracellular rhenium levels for all samples are
observed when compared to either the DMSO vehicle
treatment or the rhenium control. Cells treated with the
tridentate complex fac-[Re(CO)3(L5)]BPh4 revealed intra-
cellular rhenium concentrations of 0.236 μg per milligram of
protein. The [2 + 1] series of complexes, fac-[Re(CO)3(L1)(4-
picoline)], fac-[Re(CO)3(L1)(DMAPA)], and fac-[Re-
(CO)3(HL1)Br], show higher concentration values of 0.357,
1.30, and 2.79 μg mg−1 protein, respectively. The highest levels
of uptake are detected after treatment with fac-[Re-
(CO)3(HL1)Br], with a 119-fold increase in rhenium levels
compared with cells treated with the rhenium standard,
[Re(CO)5(CF3SO3)] (Figure 10a).
Figure 8. Excitation and emission spectra for para-nitro-bearing
complex fac-[Re(CO)3(L1)(pyridine)] emission (λex = 520 nm, light
gray) and excitation (λem = 715 nm, dark gray): 25 μM, acetonitrile.
broad maxima are assigned to 3MLCT states, which is a
characteristic excited state nature of rhenium(I) polypyridine
complexes.41,42 Though Stokes’ shifts are generally high in such
cases, the [2 + 1], hydrazone-ligand coordination compounds
presented here demonstrate larger Stokes’ values than
commonly observed (λ = 185−195 nm).
Deprotonation of the fac-[Re(CO)3(HL1)(Br)] and fac-
[Re(CO)3(HL1)(pyridine)]BPh4 complexes by the addition of
triethylamine augmented emission intensity, which indicates
that deprotonation for these complexes is not complete in
acetonitrile despite the immediate changes in color from yellow
to purple without the addition of a base.
The increased rhenium levels of fac-[Re(CO)3(HL1)Br] and
fac-[Re(CO)3(L1)(DMAPA)] may be attributed to the
increased lability of the monodentate ligating molecules,
bromine and 3-dimethylamino-1-propylamine, in comparison
to 4-picoline and pyridine. The lability of these ligands may
facilitate coordination of the rhenium to intracellular or
membrane proteins, consequently increasing the detection of
rhenium in proportion to protein levels.
The luminescence properties of such compounds may be
useful for developmental investigations into cell localization
and, due to their low energy emissions, are particularly
beneficial to distinguishing between endogenous fluorophores,
which typically emit at much higher energies.
Confocal Fluorescence Microscopy and ICP Mass
Spectrometry. The luminescence properties of these
complexes are useful for investigating cell uptake and
intracellular localization using confocal fluorescent microscopy,
as their relatively low energy excitation profiles and large
Stokes’ shifts allow images to be acquired with little
autofluorescence due to endogenous fluorophores.42−45
A proof-of-concept study identified that it was possible to
monitor cell uptake and distribution of the picoline-derived fac-
[Re(CO)3(L1)(4-picoline)] complex within a HeLa cancer cell
The [2 + 1] mixed-ligand complex fac-[Re(CO)3(L1)(4-
picoline)] and the cationic tridentate species fac-[Re-
(CO)3(L5)]+ were selected as appropriate candidates for
further hypoxia studies as they demonstrated modest but not
negligible uptake in nonhypoxic cells.
SH-(SY5Y) cells were cultivated to approximately 60%
confluency, then transferred to a sealed GasPak EZ anaerobe
container system with indicator and cultivated overnight to 80−
90% confluency before treatment to induce hypoxia-like
conditions. After the treatment solutions were prepared and
Figure 9. Confocal microscopy images of HeLa cells treated for 30 min at an excitation of λ 520 nm and with increasing concentrations of fac-
[Re(CO)3(L1)(4-picoline)] in DMSO. (a) DMSO control (0 μM), (b) 10 μM, (c) 25 μM, and (d) 50 μM.
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Figure 10. ICP-MS data of SH-(SY5Y) cells with rhenium complexes and control compounds. (a) Cells were subjected to normal oxygen levels and
treated with compound (labeled). (b) Cells were subjected to both normoxic and hypoxic conditions (GasPak EZ aerobe container).
Figure 11. HPLC chromatograms (normalized) for (a) fac-[Re(CO)3(L5)]+ (below; UV detection, λ 254 nm) and radiolabeled fac-
[
99mTc(CO)3(L5)]+ (above; γ-ray detection) and (b) fac-[Re(CO)3(L6)] (below; UV detection, λ 254 nm) and radiolabeled fac-[99mTc(CO)3(L6)]
(above; γ-ray detection).
media purged with N2, the cells were again placed in the
oxygen-deficient container with three GasPak EZ indicator
sachets and incubated at 37 °C for 1 h.
for a further 30 min. The extent of complexation was analyzed
by RP-HPLC equipped with a UV and γ-ray detector with
coinjection of cold rhenium analogues as reference compounds.
Initial attempts at translating [2 + 1] rhenium chemistry to
radioactive technetium-99m at the tracer level were marginally
successful. The 4-nitro complex fac-[99mTc(CO)3(L1)(4-pico-
line)] shows promise; however at least two radioactive species
of similar concentrations are detected in the HPLC chromato-
gram of this complex, and further method development is
required.
The standard, [Re(CO)5(CF3SO3)], again shows minimal
uptake into cells, while the tridentate fac-[Re(CO)3(L5)]+
demonstrates a 5.5-fold increase into hypoxia-induced cells
with an initial rhenium concentration of 0.409 μg mg−1 protein
compared to a concentration of 2.24 μg mg−1 protein in the
hypoxia induced cells. Surprisingly, fac-[Re(CO)3(L1)(4-pico-
line)] displays a 15.3-fold increase in cellular rhenium uptake
for the hypoxic cells (0.524 vs 8.00 μg mg−1 protein; Figure
10b), indicating the bidentate nitrobenzene complexes,
suggesting that the nitrobenzene complexes may be a viable
alternative to conventional nitroimidazole-containing systems.
Techentium-99m Radiolabeling Experiments with
HL1−2, L5 and HL6/L6Et. Radiolabeling was undertaken for
all complexes and their retention times were compared to their
rhenium analogues. The precursor fac-[99mTc(CO)3(H2O)3]+
(1000 MBq, 1 mL 0.9% saline solution) was formed using the
commercially available lyophilized IsoLink kit. Slow oxidation
of neutralized fac-[99mTc(CO)3(H2O)3]+ to [99mTcO4]− over
the duration of experimentation (4−8 h) has been previously
noted.46
Ligands L5 and either HL6 or L6Et were separately mixed
with aliquots of fac-[99mTc(CO)3(H2O)3]+ neutralized with 1.0
M HCl in a sealed vial, briefly sonicated, and heated at 65 °C
for 35 min. Preparation of the complexes fac-[99mTc-
(CO)3(L5)]+ and fac-[99mTc(CO)3(L6)] resulted in radio-
chemical yields of above 90% (Figure 11). The cationic
complex fac-[99mTc(CO)3(L5)]+ could be prepared with higher
radiochemical purity than neutral fac-[99mTc(CO)3(L6)]. The
slight difference between retention times between the 99mTc-
labeled compounds and their rhenium counterparts can, at least
in part, be attributed to the sequential configuration of the UV
and γ-ray detectors.
Evaluation of 99mTc-Labeled Tridentate Complex, fac-
Preparation of [2 + 1] mixed-ligand complexes involved
reaction of the bidentate ligands HL1 or HL2 to the
technetium-99m precursor at neutral pH with heating. The
apical monodentate ligand (pyridine or 4-picoline) was added
in excess as a 0.1 M aqueous solution (20−30 μL) and heated
[
99mTc(CO)3(L5)]+. The stability of the radiolabeled complex
fac-[99mTc(CO)3(L5)]+ was tested by challenge reactions in 0.1
M phosphate buffered saline (PBS) and human plasma at 37 °C
and analyzed by RP-HPLC at varying time intervals. Plasma
stability studies for fac-[99mTc(CO)3(L5)]+ involved the
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addition of the labeled complex in a PBS/HCl solution (0.15
mL, pH 7) to fresh human plasma from a healthy male. The
plasma was incubated at 37 °C, and 0.1 mL aliquots were
withdrawn after 10 min and 1, 2, and 4 h time intervals. No
radioactive degradation products are detected over this time
period for the radiolabeled complex.
The relative lipophilicity of the complex was determined by
the addition of 10 μL of fac-[99mTc(CO)3(L5)]+ solution
(approximately 10 MBq) to equal volumes of octanol and 0.1
M PBS, which was then shaken and separated. The radioactivity
of each phase was measured and the partition coefficient (P)
calculated (P = 0.52, cpm in octanol/cpm in PBS). The
corresponding log P(o/pbs) value of −0.28 0.03 indicates the
hydrophilic nature of the tridentate cation.
Figure 13. (a) SPECT/CT image of Mouse 3 (1100 mm3) injected
with fac-[99mTc(CO)3(L5)]+, coronal projection showing no tumor
uptake. (b) PET/MR of Mouse 3 injected with 18F-FMISO, coronal
projection showing tumor necrosis and a hypoxic rim around the
central necrotic core. Administration and scanning of 18F-FMISO
accumulation occurred 2 h post tracer injection. Arrows indicate tumor
site, and the scale bar indicates number of counts per voxel.
Preliminary SPECT imaging studies of hypoxic tumors were
undertaken in order to evaluate the feasibility of fac-
[
99mTc(CO)3(L5)]+ for future hypoxia imaging. Radiochemical
purity was greater than 90% for all imaging studies.
Three BALB/c nude mice bearing human renal cell
carcinoma (RCC; SK-RC-52) tumors were placed in supine
and injected through the tail vein with approximately 20 MBq
of fac-[99mTc(CO)3(L5)]+, and static images were acquired 2 h
post injection. It has been established that intratumoral hypoxia
exists within this cell line.47 Each mouse had different tumor
volumes: 400, 600, and 1100 mm3 for mouse 1, 2, and 3,
respectively. Tumor uptake is not evident for the 400 mm3
(Mouse 1) or 1100 mm3 (Mouse 3) tumor models. Significant
tumor accumulation is visualized for the animal with a tumor
volume of 600 mm3, with a maximum standardized uptake
value (SUVmax) of 6.4 and approximately 1.44%ID/g tissue on
the basis of SPECT quantification (Figure 12). It is possible
(CO)3(L1−4)(X)] (X = 4-picoline, pyridine, DMAPA) were
synthesized by microwave-assisted thermal heating with short
reaction times to give products in good yields and high purity.
These complexes were characterized by mass spectroscopy,
NMR, IR, UV−visible and fluorescence spectroscopy, and X-
ray crystallography. Electrochemical studies investigated the
relative inductive influences and resonance effects of the
assorted ligand substituents (H, OCH3, m-NO2, p-NO2), in
addition to highlighting the complicated mechanism of
reduction of the nitrobenzene-containing species. Cyclic
voltammetry reveals reductive processes for the nitro-bearing
rhenium complexes with E0′ values between −0.6 and −1.1 V
that are within biologically accessible electrochemical poten-
tials. Crystallographic studies show that ligands HL1−2
crystallize as their E isomer around the CN bond. Crystal
structures of complexes with apical bromo-ligand substitution,
fac-[Re(CO)3(HL1−4)(Br)], confirm the assumptions based on
NMR data, suggesting that the bidentate hydrazine ligand
remains protonated at the hydrazinic NH following coordina-
tion. Upon exchange of the apical, monodentate ligand with N-
heterocyclic compounds, pyridine or picoline, the hydrazinic
nitrogen atom is deprotonated, inducing interesting visible
spectroscopic changes.
Two tridentate ligands bearing a nitroimidazole functional
group, L5 and HL6, were synthesized and used to prepare
cationic fac-[Re(CO)3(L5)]+ and neutral fac-[Re(CO)3(L6)].
Crystallographic analysis of fac-[M(CO)3(L5)]+ shows a
predictable six-coordinate distorted octahedral geometry with
a N,N,N ligand binding mode upon complexation. Electro-
chemical analysis using cyclic voltammetry indicates reduction
of the nitro group in the cathodic region of interest for
biological hypoxia detection for both fac-[Re(CO)3(L5)]+ and
fac-[Re(CO)3(L6)].
Figure 12. SPECT-CT images of fac-[99mTc(CO)3(L5)]+, 2 h p.i. (a)
Mouse 2 (600 mm3); (i) coronal projection, (ii) sagittal plane. (b)
Mouse 1 (400 mm3); (i) coronal projection, (ii) sagittal plane. Arrows
indicate tumor site. Scale bar indicates number of counts per voxel.
that the small tumor in Mouse 1 was too small for significant
hypoxia, but this was not verified. A separate PET/CT study of
the Mouse 3 (1100 mm3) with the hypoxia tracer 18F-FMISO
also revealed little penetration of the tracer into the tumor but a
small amount of uptake of the tracer around the outer edge of
the necrotic core (Figure 13). The SK-RC-52 tumor model has
been well characterized, and tumor size does serve as a useful
guide to tumor hypoxia, but it is acknowledged that hypoxia
cannot be predicted by tumor size alone.47−49 The promising
results from this pilot study suggest that fac-[99mTc(CO)3(L5)]+
warrants further attention in more detailed studies that include
parallel oxygen determination measurements.
The cellular uptake and retention of the [2 + 1] complex fac-
[Re(CO)3(L1)(4-picoline)] and the cationic complex prepared
with the neutral tridentate ligand fac-[Re(CO)3(L5)]+ was
investigated in normoxic and hypoxic SH-(SY5Y) cells. The
cationic complex fac-[Re(CO)3(L5)]+ has a 5-fold increase in
retention in hypoxia-induced cells, and fac-[Re(CO)3(L1)(4-
picoline)] displays a 15-fold increase in hypoxic cells.
CONCLUDING REMARKS
■
A series of ligands (HL1−4) and their matching, neutral rhenium
complexes fac-[Re(CO)3(HL1−4)(Br)] and fac-[Re-
Forming complexes with high radiochemical purity was more
straightforward with the tridentate ligands, L5 and HL6, than
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of measurement. Melting points were determined using an EZ-Melt
MPA 120 Automated Melting Point Apparatus from Stanford
Research Systems and are uncorrected.
with the nitro-containing [2 + 1] mixed-ligand systems.
Complex fac-[99mTc(CO)3(L5)]+ is stable in human serum
and relatively hydrophilic, yet it is still cell permeable (log
P(o/pbs) = −0.28). Preliminary SPECT/CT imaging with fac-
Analytical radio-HPLC was performed using a Shimadzu 10 AVP
UV−visible spectrophotometer (Shimadzu, Kyoto, Japan) with two
LC-10ATVP solvent delivery systems for solvents A and B. Peak
separation was achieved using a Nacalai Tesque Cosomosil 5C18-AR
Waters column (4.6 × 150 mm, 5 μm; Nacalai Tesque, Kyoto, Japan)
at 1 mL min−1. System C: Gradient elution of buffer A (0.1% TFA in
H2O) and buffer B (0.1% TFA in CH3CN) from 0 to 100% B to A,
over 20 min and UV detection at λ 254 nm.
Human blood samples were centrifuged with a Heraeus Labofuge
6000 centrifuge at 3000g for 10 min (Heraeus, Hanau, Germany).
Radioactivity readings for serum stability studies were taken with a
Capintec CRC-35R dose calibrator (Capintec, New Jersey, USA) and
were measured in MBq.
Centrifugation of radioactive compounds was undertaken using an
Eppendorf 5415 D centrifuge (Eppendorf, Hamburg, Germany).
Partition coefficient data were collected with a PerkinElmer, Wizard
1470 (PerkinElmer, Massachusetts, USA) automatic γ counter, which
measured the radioactive decay of each sample in counts per minute
(cpm).
SPECT images were obtained using a Mediso nanoScan SPECT/
CT (and MRI subsystem; Mediso Medical Imaging Systems, Budapest,
Hungary) equipped with a multipinhole (M3) collimator. Visualization
and evaluation of SPECT data was carried out using PMOD fusion
software (PMOD technologies, Switzerland). Organ uptake was
initially expressed as counts per voxel and then calculated as the
percentage of injected dose (%ID) and percentage of injected dose per
gram (%ID/g) volume. Animals were imaged at the Australian Cancer
Research Foundation (ACRF) Centre for Translational Cancer
Therapeutics and Imaging.
[
99mTc(CO)3(L5)]+ in a well characterized model of tumor
hypoxia revealed significant tumor accumulation (SUVmax = 6.4,
1.44%ID/g), and this complex warrants further investigation in
more detailed hypoxia models. Ligands L5 and L6 also form
stable complexes with the fac-{Re(CO)3}+ core, so inves-
tigations into radiolabeling with the therapeutic β-emitting
186/188Re isotopes are warranted with a view to developing
complexes suitable for both diagnostic imaging and therapy.
EXPERIMENTAL SECTION
■
General Procedures. All reagents and solvents, unless otherwise
stated, were purchased from standard commercial sources and were
used as received. Anhydrous ethanol (EtOH) was dried over
magnesium ethoxide under a nitrogen environment. N,N-dimethylfor-
mamide (DMF) was distilled and dried over magnesium sulfate and
stored over 4 Å molecular sieves. Acetonitrile (CH3CN) was dried
over K2CO3, distilled, and stored over 3 Å molecular sieves. All
reactions were undertaken under inert conditions unless otherwise
indicated. A Biotage Initiator Sixty EXP 2.0 Microwave Reactor was
utilized for all microwave-assisted reactions. Thin-layer chromatog-
raphy (TLC) was performed on Merck aluminum backed 2-μm-thick
silica gel plates (60 GF254) and were visualized under λ 254 nm UV
light or stained with 20% w/v phosphomolybdic acid in ethanol, iodine
dip, or 20% w/v copper chloride in acetone where relevant. Column
chromatography was performed on silica gel (Kieselgel 60, 230−400
mesh) using the indicated solvent system. Nuclear Magnetic
Resonance (NMR) spectra were acquired on a superconducting
Varian-FT-NMR 500 spectrometer (Varian, California, USA). All
Syntheses. [Re(CO)5OTf],50 1H-imidazol-2-amine sulfate,51
potassium 2-nitro-1H-imidazolide,37 1-(3-bromopropyl)-2-nitro-1H-
imidazole,37 and N-(2-pyridylmethyl)aminoethyl acetate52 were
synthesized according to previously reported procedures. Ligands
HL1 and HL2 are known compounds and their spectroscopic details
are available from the literature;53,54 however NMR spectral data and
melting points vary significantly from values obtained for this research,
therefore full spectral analysis has been provided.
1
NMR were acquired at 25 °C. H and 13C{1H} chemical shifts were
referenced to residual solvent peaks and are quoted in parts per million
relative to TMS. Mass Spectra (MS) were recorded on an Agilent 6220
Q-TOF LC/MS (Agilent, California, USA) mass spectrometer and/or
a Waters-Micromass QuattroII triple quadrupole electrospray
ionization mass spectrometer. Cyclic voltammograms were recorded
on an AUTOLAB PGSTAT100 electrochemical workstation using
GPES V4.9 software. A glassy-carbon working electrode, platinum wire
counter electrode, and Ag/Ag+ (silver wire immersed in 0.01 M
AgNO3/CH3CN) reference electrode were utilized for all measure-
ments. Measurements were taken in deoxygenated, anhydrous CH3CN
with 0.1 M supporting electrolyte, [NH4][BF4], and 1.0 mM analyte
unless otherwise specified. Each solution was measured at ambient
temperature, and the cell was kept under a N2 atmosphere throughout
the experiment. Cathodic peak potentials (Epc) and half-wave potential
(E0′) were measured relative to an internal reference ferrocenium/
ferrocene (Fc+/Fc) redox couple, where E0′ was designated as +400
mV vs a saturated calomel electrode (SCE) in acetonitrile.31
Analytical Reverse Phase High Performance Liquid Chromatog-
raphy (RP-HPLC) was undertaken using an Agilent 1100 Series
HPLC System using a Zorbax Eclipse XDB-C18 column (4.6 × 150
mm, 5 μm) at a flow rate of 1 mL min−1. System A: Gradient elution
of buffer A (0.1% TFA in H2O) and buffer B (0.1% TFA in CH3CN)
from 0 to 100% B to A over 25 min and UV detection at λ 214, 254,
and 280 nm. System B: Gradient elution of buffer A (0.1% TFA in
H2O) and buffer B (0.1% TFA in CH3CN) from 0 to 40% B to A over
25 min and UV detection at λ 214, 254, and 280 nm.
HL1. To 2-hydrazinopyridine (3.00 g, 27 mmol) in ethanol (40 mL)
was added 4-nitrobenzaldehyde (4.15 g, 27 mmol). The resulting
suspension was stirred for 4 h at RT. The precipitate was collected via
vacuum filtration and washed with cold ethanol (3 × 10 mL) and
diethyl ether (3 × 10 mL). The solid was purified by column
chromatography (CH2Cl2/CH3OH, 95:5), and the desired fractions
were recrystallized from hot dichloromethane to give orange stick
needles (5.02 g, 77%). 1H NMR [DMSO-d6, 400 MHz]: δ (ppm) 11.3
(1H, s, NH), 8.23 (2H, AA′XX′, ArH), 8.16 (1H, d, J = 4.4 Hz, pyH),
8.09 (1H, s, CHN), 7.90 (2H, AA′XX′, ArH), 7.69 (1H, t, J = 7.6
Hz, pyH), 7.32 (1H, d, J = 8.4 Hz, pyH) 6.84 (1H, t, J = 6.0 Hz, pyH).
13C{1H} NMR [DMSO-d6, 125.7 MHz]: δ (ppm) 156.5 (C, pyC),
147.9 (C, ArC-NO2), 146.5 (C, pyC), 142.0 (C, CHN), 138.1 (C,
ArC), 136.0 (C, pyCH), 126.5 (2C, ArCH), 124.0 (2C, ArCH), 115.9
(C, pyCH), 106.8 (C, pyCH). IR: ν (cm−1) 2987 (Ar C−H), 1504
(ArNO2, νas(NO)2) 1333 (ArNO2, νsym(NO)2). HRMS (ESI+)
m/z calcd for C12H11N4O2: 243.0882. Found: 243.0877 {[M + H]+,
100%}. mp: 231−237 °C. Crystals suitable for single crystal X-ray
crystallography were grown from a concentrated solution in
dimethysulfoxide.
HL2. The procedure is as that for HL1, except using 2-
hydrazinopyridine (3.00 g, 27 mmol), ethanol (40 mL), and 3-
nitrobenzaldehyde (4.15 g, 27 mmol). The solid was recrystallized
UV−visible absorption spectra were recorded on a Shimadzu UV-
1650PC UV−visible spectrophotometer using UVPC v3.9 software.
Emission spectra were obtained on a Varian CARY Eclipse
fluorescence spectrophotometer. Both absorption and emission
spectra measurements were performed in capped quartz cuvettes in
THF or CH3CN.
Infrared spectra (IR) were recorded neat on a PerkinElmer
Spectrum One FT-IR spectrometer with a zinc selenide/diamond
universal ATR 60 sampling accessory, as a thin film using cm−1 as units
1
from hot chloroform to afford a yellow fluffy solid (4.81 g, 74%). H
NMR [CH3CN-d3, 500 MHz]: δ (ppm) 10.4 (1H, s, NH), 8.47 (1H, s,
ArH), 8.13−8.10 (2H, m, ArH), 8.07 (1H, s, CHN), 8.04 (1H, dtd,
J = 7.8, 1.3, 0.5 Hz, pyH), 7.65 (1H, ddd, J = 8.4, 1.2, 1.9 Hz, pyH),
7.61 (1H, t, J = 8.0 Hz, ArH), 7.33 (1H, dt, J = 8.4, 1.0 Hz, pyH), 6.80
(1H, ddd, J = 7.2, 4.9, 1.0 Hz, pyH). 13C{1H} NMR [DMSO-d6, 125.7
MHz]: δ (ppm) 156.7 (C, pyC), 148.3 (C, ArC-NO2), 147.8 (C,
L
DOI: 10.1021/acs.inorgchem.5b01691
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Inorganic Chemistry
Article
pyCH), 138.1 (C, NCH), 137.3 (C, ArC), 136.2 (C, pyC), 132.0
(C, ArCH), 130.3 (C, ArCH), 122.6 (C, ArCH), 119.8 (C, ArCH),
115.6 (C, pyCH), 106.6 (C, pyCH). IR: ν (cm−1) 2972−2901 (Ar C−
H), 1520 (ArNO2, νas(NO)2), 1343 (ArNO2, νsym(NO)2).
HRMS (ESI+) m/z calcd for C12H11N4O2: 243.0882. Found:
243.0877 {[M + H]+, 100%}; mp 220−228 °C. Crystals suitable for
single crystal X-ray crystallography were grown from a concentrated
solution in dimethyl sulfoxide.
fac-[Re(CO)3(4-picoline)3]OTf. This compound was prepared by a
modification of a previously reported procedure.56 Freshly distilled 4-
p i c o l i n e ( 2 m L ) w a s a d d e d t o p e n t a c a r b o n y l -
(trifluoromethanesulfonato) rhenium(I) (0.16 g, 0.33 mmol) and
subjected to microwave conditions at 150 °C for 15 min (×2). The
solution was cooled to RT, and diethyl ether (6 mL) was added. The
resulting precipitate was filtered and washed with cold diethyl ether to
afford a white powder (0.18 g, 78%). 1H NMR [CH3CN-d3, 500
MHz]: δ (ppm) 8.31 (6H, AA′XX′, picH), 7.36 (6H, AA′XX′, picH),
2.44 (9H, s, CH3). 13C{1H} NMR [CH3CN-d3, 125.7 MHz]: δ (ppm)
154.1 (3C, ArC), 153.9 (6C, ArCH), 128.7 (6C, ArCH), 21.30 (9C,
CH3). IR: ν (cm−1) 2027 (fac-ν(CO)), 1903 (fac-ν(CO)).
HRMS (ESI+) m/z calcd for C21H21N3O3Re: 550.1140. Found:
550.1142 {[M-OTf]+, 100%}. RP-HPLC (System A): RT (min) 14.0
fac-[Re(CO)3(DMAPA)3]2OTf. This compound was prepared by a
modification of a previously reported procedure56 but was not isolated;
formation was determined by ESI-MS. ESI-MS calcd for
C18H42N6O3Re m/z 577.2876. Found: 577.2888 {[M-2OTf]+, 100%}
fac-[Re(CO)3(HL1)Br]. Rhenium triaquatricarbonyl bromide (0.1 g,
0.25 mmol) was stirred in anhydrous methanol (10 mL). HL1(0.06 g,
0.25 mmol) was added to the solution under an inert atmosphere and
stirred at RT for 4 h. The resulting precipitate was collected via
vacuum filtration and washed with cold methanol. A yellow solid was
obtained as fac-[Re(CO)31(HL1)Br] (0.09 g, 60%) which turned a deep
pink/purple in solution. H NMR [DMSO-d6, 500 MHz]: δ (ppm)
13.2 (1H, br s, NH), 8.67 (1H, s, CHN), 8.42 (2H, AA′XX′, ArH),
8.36 (1H, d, J = 5.2 Hz, pyH), 7.99 (2H, AA′XX′, ArH), 7.91 (1H, tdd,
J = 7.5, 7.0, 1.5 Hz, pyH), 7.12 (1H, d, J = 8.5 Hz, pyH), 6.98 (1H, t, J
= 6.5 Hz, pyH). 13C{1H} NMR [DMSO-d6, 125.7 MHz]: δ (ppm)
196.6 (C, CO), 196.5 (C, CO), 194.6 (C, CO), 155.1 (C, pyCH),
151.3, 149.5, 148.3 (C, ArC-NO2), 140.8, 140.2, 131.0 (2C, ArCH),
123.6 (2C, ArCH), 117.2 (C, pyCH), 108.2 (C, pyCH). IR: ν (cm−1)
2988 (Ar C−H, N−H), 2022 (fac-ν(CO)), 1894 (fac-ν(CO)),
1522 (ArNO2, νas(NO)2) 1357 (ArNO2, νsym(NO)2). HRMS
(ESI−) m/z calcd for C15H9BrN4O5Re: 590.9314. Found: 590.9314
{[M−H]−, 100%}. RP-HPLC (System A): RT (min) 15.31. Crystals
suitable for single crystal X-ray crystallography were grown from slow
evaporation of a concentrated solution in acetonitrile.
HL3 (+ 1/4 H2O). The procedure is as that for HL1, except using 2-
hydrazinopyridine (0.81 g, 7.4 mmol), ethanol (10 mL), and 4-
methoxybenzaldehyde (0.89 mL, 7.4 mmol). The collected precipitate
was sufficiently clean to proceed without further purification and was
1
obtained as a white fluffy solid (1.20 g, 72%). H NMR [DMSO-d6,
500 MHz]: δ (ppm) 10.7 (1H, s, NH), 8.08 (1H, ddd, J = 4.9, 1.9, 0.9
Hz, pyH), 7.98 (1H, s, CHN), 7.63−7.59 (3H, m, pyH, ArH, ArH),
7.21 (1H, dt, J = 8.4, 0.9 Hz, pyH), 6.97 (2H, AA′XX′, ArH), 6.72
(1H, ddd, J = 7.1, 4.9, 1.0 Hz, pyH), 3.78 (3H, s, CH3). 13C{1H} NMR
[DMSO-d6, 125.7 MHz]: δ (ppm) 159.7, 157.2, 147.7, 138.8, 137.8,
128.1 (2C, ArCH), 127.3 (C, ArC), 114.5 (C, pyCH), 114.2 (2C,
ArCH), 106.1 (C, pyCH), 55.2 (C, OCH3). IR: ν (cm−1) 3180−2836
(Ar/py C−H), 1596 (CC, CN), 1438 (CC, CN), 1244
(OCH3, νas(C−O−C)), 1036 (OMe, νsym(C−O−C)). HRMS (ESI+)
m/z calcd for C13H14N3O: 227.11369. Found: 227.1141 {[M + H]+,
100%}. mp: 165−167 °C.
HL4 (+ 1/4 H2O). The procedure is as that for HL1, except using 2-
hydrazinopyridine (0.80 g, 7.1 mmol), ethanol (10 mL), and
benzaldehyde (0.82 mL, 7.1 mmol). The collected precipitate was
sufficiently clean to proceed without further purification and was
1
obtained as a pale yellow solid (1.0 g, 72%). H NMR [CDCl3, 500
MHz]: δ (ppm) 10.9 (1H, s, NH), 8.11 (1H, ddd, J = 4.9, 1.9, 0.9 Hz,
pyH), 8.03 (1H, s, CHN), 7.67−7.62 (3H, m, ArH, ArH, pyH), 7.40
(2H, dd, J = 8.0, 6.9 Hz, ArH), 7.32 (1H, tt, J = 7.0, 1.5 Hz), 7.25 (1H,
dt, J = 8.4, 0.9 Hz), 6.79 (1H, ddd, J = 7.1, 4.9, 1.0 Hz, pyH). 13C{1H}
NMR [CDCl3, 125.7 MHz]: δ (ppm) 156.7, 147.0, 139.7, 138.6,
135.0, 129.0 (2C, ArCH), 126.6 (2C, ArCH), 115.8 (C, pyCH), 107.9
(C, pyCH). IR: ν (cm−1) 3195−2901 (Ar/py C−H), 1603 (CC,
CN), 1436 (CC, CN). HRMS (ESI+) m/z calcd for
C12H11N4O2: 198.1031. Found: 198.1032 {[M + H]+, 100%}. mp:
150−152 °C.
fac-[Re(CO)3(HL2)Br]. This procedure is as per that for fac-
[Re(CO)3(HL1)Br] except using HL2 as the ligand. A pale yellow
fac-[Re(H2O)3(CO)3]Br. This compound was prepared by a
modification of a previously reported procedure.55 To bromopenta-
carbonyl rhenium(I) (0.40 g, 0.98 mmol) was added degassed Milli-Q
water (10 mL). The suspension was subjected to microwave heating
for 10 min at 150 °C. The reaction mixture was vigorously shaken then
resubjected under the same conditions (×2). The solution was filtered
hot and reduced in vacuo to afford a pale green powder (0.39 g, 98%).
IR: ν (cm−1) 2018 (fac-ν(CO)), 1910 (fac-ν(CO)).
1
powder was obtained as fac-[Re(CO)3(HL2)Br] (0.12 g, 80%). H
NMR [DMSO-d6, 500 MHz]: δ (ppm) 13.2 (1H, s, NH), 8.71 (1H, s,
ArH), 8.66 (1H, s, CHN), 8.41 (1H, d, J = 8 Hz, ArH), 8.36 (1H, d,
J = 4.5 Hz, ArH), 8.17 (1H, d, J = 7.5 Hz, pyH), 7.88 (2H, m, ArH,
pyH), 7.11 (1H, d, J = 8.5 Hz, pyH), 6.97 (1H, t, J = 5.5 Hz, pyH).
13C{1H} NMR [DMSO-d6, 125.7 MHz]: δ (ppm) 196.1 (C, CO)
194.8 (C, CO), 190.5 (C, CO), 155.2 (C, pyC), 150.9 (C, NCH),
149.4 (C, pyCH), 147.5 (C, C-NO2), 140.7, 135.9, 135.3 (C, ArC),
130.3, 125.0, 124.6 (C, ArCH), 117.1 (C, pyCH), 108.1 (C, pyCH).
IR: ν (cm−1) 3184−3051 (ν(N−H)), 2023 (fac-ν(CO)), 1902 (fac-
ν(CO)), 1519 (ArNO2, νas(NO)2), 1344 (ArNO2, νsym(N
O)2). HRMS (ESI+) m/z calcd for C15H10N4O5Re: 513.0203. Found:
513.0203 {[M−Br]+, 100%}. RP-HPLC (System A): RT (min) 14.2.
Crystals suitable for single crystal X-ray crystallography were grown
from slow evaporation of a concentrated solution in acetonitrile.
fac-[Re(CO)3(HL3)Br]. This procedure is as per that for fac-
[Re(CO)3(HL1)Br] except using HL3 (0.06 g, 0.25 mmol) as the
ligand. An off-white powder was obtained as fac-[Re(CO)3(HL3)Br]
(0.07 g, 49%). 1H NMR [DMSO-d6, 500 MHz]: δ (ppm) 12.8 (1H, br
s, NH), 8.52 (1H, s, CHN), 8.34 (1H, ddd, J = 5.9, 1.6, 0.8 Hz,
pyH), 7.86 (1H, t, J = 7.0 Hz, pyH), 7.81 (2H, AA′XX′, ArH), 7.12
(2H, AA′XX′, ArH), 7.05 (1H, dt, J = 8.5, 0.9 Hz, pyH), 6.92 (1H,
ddd, J = 7.2, 5.9, 1.2 Hz pyH). 13C{1H} NMR [DMSO-d6, 125.7
MHz]: δ (ppm) 196.4 (C, CO), 194.9 (C, CO), 191.3 (C, CO), 161.5
(C, ArC-OCH3), 155.4 (C, pyC), 153.4 (C, NCH), 149.3 (C,
pyCH), 140.3 (C, pyCH), 131.7 (2C, ArCH), 125.6 (C, ArC), 116.4
(C, pyCH), 114.0 (2C, ArCH), 107.9 (C, pyCH), 55.5 (C, OCH3). IR:
ν (cm−1) 3182−2931 (ν(N−H)), 2020 (fac-ν(CO)), 1916 (fac-
ν(CO)1878 (fac-ν(CO)), 1605 (CC, CN), 1484 (CC,
CN), 1263 (OCH3, νas(C−O−C)), 1176 (OCH3, νsym(C−O−C)).
fac-[Re(H2O)3(CO)3]OTf. This compound was prepared by a
modification of a previously reported procedure.55,56 To
pentacarbonyl(trifluoromethanesulfonato) rhenium(I) (0.30 g, 0.63
mmol) was added degassed Milli-Q water (6.3 mL). The suspension
was subjected to microwave heating for 15 min at 155 °C. The
reaction mixture was stored as a 0.1 M solution (approx.) in water.
fac-[Re(CO)3(pyridine)3]OTf. This compound was prepared by a
modification of a previously reported procedure.56 To pentacarbonyl-
(trifluoromethanesulfonato) rhenium(I) (0.19 g, 0.31 mmol) purged
with N2 was added degassed pyridine (4 mL). The solution was
subjected to microwave conditions at 150 °C for 15 min. The pyridine
was reduced to approximately 1/4 under a high vacuum and diethyl
ether (4 mL) added and stirred vigorously, whereby a white solid
precipitated. The solid was filtered and washed with copious amounts
1
of cold diethyl ether to afford white crystals (0.22 g, 83%). H NMR
[CH3CN-d3, 500 MHz]: δ (ppm) 8.51 (6H, d, J = 6.0 Hz, pyH), 8.10
(3H, t, J = 7.5 Hz, pyH), 7.54 (6H, t, J = 7.0 Hz, pyH). 13C{1H} NMR
[CH3CN-d3, 125.7 MHz]: δ (ppm) 154.7 (6C, ArCH), 141.4 (6C,
ArCH), 128.1 (3C, ArCH). IR: ν (cm−1) 2027 (fac-ν(CO)), 1908
(fac-ν(CO)). HRMS (ESI+) m/z calcd for C18H15N3O3Re:
508.0671. Found: 508.0680 {[M-OTf]+, 100%}. RP-HPLC (System
A): RT (min) 11.9.
M
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HRMS (ESI+) m/z calcd for C16H13N3O4Re: 498.0464. Found:
498.0470 {[M−Br]+, 100%}. RP-HPLC (System A): RT (min) 15.0.
Crystals suitable for single crystal X-ray crystallography were grown
from slow evaporation of a concentrated solution in acetonitrile.
fac-[Re(CO)3(HL4)Br]. This procedure is as per that for fac-
[Re(CO)3(HL1)Br] except using HL4 (0.05 g, 0.25 mmol) as the
ligand. A pale yellow powder was afforded as fac-[Re(CO)3(HL4)Br]
(0.09 g, 69%). 1H NMR [DMSO-d6, 500 MHz]: δ (ppm) 12.9 (1H, br
s, NH), 8.61 (1H, s, CHN), 8.34 (1H, ddd, J = 5.8, 1.6, 0.8 Hz,
pyH), 7.88 (1H, ddd, J = 8.6, 7.2, 1.5 Hz, pyH), 7.77−7.75 (2H, m,
ArH), 7.56−7.55 (3H, m, ArH), 7.07 (1H, dt, J = 8.5, 1.0 Hz, pyH),
6.94 (1H, ddd, J = 7.2, 5.9. 1.2, pyH). 13C{1H} NMR [DMSO-d6,
125.7 MHz]: δ (ppm) 196.4 (C, CO) 194.4 (C, CO), 191.1 (C, CO),
155.3 (C, pyC), 153.3 (C, NCH), 149.4 (C, pyCH), 140.4, 133.6
(C, ArC), 130.7, 129.4 (2C, ArCH), 128.5 (2C, ArCH), 116.6 (C,
pyCH), 107.9 (C, pyCH). IR: ν (cm−1) 3180−3020 (ν(N−H)), 2020
(fac-ν(CO)), 1921 (fac-ν(CO)), 1885 (fac-ν(CO)), 1626−
1427 (py C−N, C−C). HRMS (ESI+) m/z calcd for C15H11N3O3Re:
468.0358. Found: 468.0440 {[M-Br]+, 100%}. RP-HPLC (System A):
RT (min) 15.2. Crystals suitable for single crystal X-ray crystallography
were grown from slow evaporation of a concentrated solution in
acetonitrile.
fac-[Re(CO)3(HL1)(pyridine)]BPh4. This proceure is as that for fac-
[Re(CO)3(L1)(4-picoline)], except using HL1 (0.04 g, 0.15 mmol),
fac-[Re(CO)3(pyridine)3]OTf (0.1 g, 0.15 mmol), and CH3OH (4
mL). Analytical data refer to the deprotonated complex fac-
[Re(CO)3(L1)(pyridine)] with no counterion present. 1H NMR
[CH3CN-d3, 500 MHz]: δ (ppm) 8.65 (2H, AA′XX′, ArH), 8.50−8.49
(2H, m, pyHan), 8.35 (1H, ddd, J = 6.0, 1.7, 0.9 Hz, pyH), 8.21 (2H,
AA′XX′, ArH), 7.89−7.86 (1H, m, pyHan), 7.88 (1H, s CHN), 7.43
(1H, ddd, J = 8.7, 6.9, 1.8 Hz, pyH), 7.36−7.33 (2H, m, pyHan), 6.90
(1H, dt, J = 8.7, 1.1 Hz, pyH), 6.43 (1H, ddd, J = 6.9, 5.9, 1.2 Hz,
pyH). 13C{1H} NMR [CH3CN-d3, 125.7 MHz]: δ (ppm) 198.8 (C,
CO), 198.7 (C, CO), 195.6 (C, CO), 170.3 (C, pyC), 152.9 (2C,
pyCHan), 149.1 (C, pyCH), 141.5, 140.4, 139.2 (C, pyCHan), 132.1
(2C, ArCH), 127.1 (2C, pyCHan), 124.2 (2C, ArCH), 114.8 (2C,
pyCH), 113.2 (2C, pyCH). IR: ν (cm−1) 2034 (fac-ν(CO)), 1933
(fac-ν(CO)), 1902 (fac-ν(CO)), 1523 (ArNO2, νas(NO)2).
1342 (ArNO2, νsym(NO)2). HRMS (ESI+) m/z calcd for
C20H15N5O5Re: 592.0631. Found: 592.0692 {[M + H]+, 100%}. RP-
HPLC (System A): RT (min) 13.4. Yellow block crystals suitable for
X-ray crystallography were grown by the addition of excess NaBPh4 to
a concentrated solution of fac-[Re(CO)3(L1)(pyridine)] in CH3OH
followed by cooling at 0 °C for 3 days.
fac-[Re(CO)3(L1)(4-picoline)]. Degassed, anhydrous CH3OH (2
mL) was added to HL1 (0.04 g, 0.14 mmol). The tris-substituted
rhenium complex fac-[Re(CO)3(4-picoline)3]OTf (0.1 g, 0.14 mmol)
was dissolved in degassed, anhydrous CH3OH (3 mL) and added to
the suspension. The reaction mixture was subjected to microwave
irradiation at 155 °C for 2 × 10 min. The solution was cooled, and the
resultant precipitate was collected via vacuum filtration. The filtrate
was collected and reduced in vacuo to approximately 1/2 the initial
volume, followed by the addition of Et2O to yield a second crop of the
desired product. A forest-green, crystalline solid was obtained (0.07 g,
80%). 1H NMR [CH3CN-d3, 500 MHz]: δ (ppm) 8.65 (2H, AA′XX′,
ArH), 8.35 (1H, dd, J = 6.0, 0.6 Hz, pyH), 8.32 (2H, d, J = 5.5 Hz,
picH), 8.21 (2H, AA′XX′, ArH), 7.87 (1H, s, ArH), 7.45−7.42 (1H, t,
J = 8.0 Hz, pyH), 7.17 (2H, dd, J = 5.9, 0.4 Hz, picH), 6.90 (1H, d, J =
8.7 Hz, pyH), 6.42 (1H, t, J = 6.4 Hz, pyH), 2.30 (3H, s, CH3).
13C{1H} NMR [CH3CN-d3, 125.7 MHz]: δ (ppm) 199.0 (C, CO)
198.9 (C, CO), 195.7 (C, CO), 170.5 (C, pyC), 153.1 (C, picCan),
152.2 (2C, picCHan), 149.2 (C, pyCH), 147.2 (C, C-NO2), 141.5 (C,
NCH), 140.5 (C, ArC), 139.3 (C, ArCH), 132.2 (2C, ArCH), 128.0
(2C, picCHan), 124.3 (2C, ArCH), 114.9 (C, pyCH), 113.3 (C,
pyCH), 21.11 (C, picCH3). IR: ν (cm−1) 2016 (fac-ν(CO)), 1929
(fac-ν(CO)), 1897 (fac-ν(CO)), 1505 (ArNO2, νas(NO)2)
1332 (ArNO2, νsym(NO)2). HRMS (ESI+) m/z calcd for
C21H17N5O5Re: 606.0787. Found: 606.0805 {[M + H]+, 100%}. RP-
HPLC (System A): RT (min) 14.2. Crystals suitable for single-crystal
X-ray crystallography were grown from slow evaporation of a
concentrated solution of the product in acetonitrile.
fac-[Re(CO)3(L2)(pyridine)]. This proceure is as that for fac-
[Re(CO)3(L1)(4-picoline)], except using L2 (0.04 g, 0.15 mmol),
fac-[Re(CO)3(pyridine)3]OTf (0.1 g, 0.15 mmol), and CH3OH (4
mL). Bright orange crystals were afforded as the desired product (1st
crop) or orange powder (2nd crop; 0.06 g, 69%, total yield). 1H NMR
[CH3CN-d3, 500 MHz]: δ (ppm) 9.64 (1H, s, CHN), 8.58 (1H, d, J
= 7.9 Hz), 8.51 (2H, AA′XX′, pyH), 8.33 (1H, dd, J = 5.9. 0.9 Hz,
pyH), 8.14 (1H, ddd, J = 8.2, 2.3, 0.9 Hz, pyH), 7.92 (1H, s, pyH),
7.88 (1H, tt, J = 7.7, 1.6 Hz, pyH), 7.63 (1H, t, J = 8.0 Hz, pyH), 7.41
(1H, ddd, J = 8.6, 6.9, 1.7 Hz), 7.37−7.34 (2H, AA′XX′, ArH), 6.85
(1H, d, J = 8.7 Hz, pyH). 13C{1H} NMR [CH3CN-d6, 125.7 MHz]: δ
(ppm) 198.9 (2C, CO), 195.9 (C, CO), 153.0 (2C, pyCHan), 149.2
(C, ArC-NO2), 149.1 (C, PyCH), 142.1 (C, NCH), 140.5 (C,
pyCHan), 139.1 (C, PyCH), 137.5 (C, ArCH), 136.3 (C, ArC), 130.2
(C, ArCH), 127.2 (2C, pyCHan), 126.2 (C, ArCH), 123.9 (C, ArCH),
114.4 (C, pyCH), 112.7 (C, pyCH). IR: ν (cm−1) 2016 (fac-ν(C
O)), 1893 (fac-ν(CO)), 1524 (ArNO2, νas(NO)2), 1351
(ArNO2, νsym(NO)2). HRMS (ESI+) m/z calcd for
C20H15N5O5Re 592.0631. Found: 592.0635 {[M + H]+, 100%}. RP-
HPLC (System A): RT (min) 13.5. Crystals suitable for X-ray
crystallography were grown from a concentrated solution of methanol.
fac-[Re(CO)3(L1)(N,N′-dimethylaminopropylamine)]. This pro-
ceure is as that for fac-[Re(CO)3(L1)(4-picoline)], except using L1
(0.03 g, 0.12 mmol) in hot CH3OH (1 mL) and fac-[Re-
(CO)3(DMAPA)3]2+ (approximately 0.12 mmol) in a 1:1 CH3OH/
DMAPA solution. Kugelrohr distillation was used to remove excess
DMAPA. Dark red powder was obtained to afford the desired product
1
fac-[Re(CO)3(L2)(4-picoline)]. This proceure is as that for fac-
[Re(CO)3(4-picoline)3L1], except using HL2 (0.04 g, 0.14 mmol), fac-
[Re(CO)3(4-picoline)3]OTf (0.10 g, 0.14 mmol), and CH3OH (4
mL). Bright orange crystals were obtained from the reaction mixture
(0.06 g, 69%). 1H NMR [CH3CN-d3, 500 MHz]: δ (ppm) 9.66 (1H, s,
CHN), 8.58 (1H, d, J = 7.9 Hz, ArH), 8.34−8.30 (3H, m, picH,
pyH), 8.13 (1H, d, J = 7.7 Hz, ArH), 7.89 (1H, s, ArH), 7.62 (1H, td, J
= 8.1, 1.5 Hz, ArH), 7.39 (1H, ddt, J = 8.7, 6.9, 1.8 Hz, pyH), 7.17
(2H, d, J = 5.4 Hz, picH), 6.83 (1H, d, J = 8.7 Hz, pyH), 6.36 (1H, t, J
= 6.4 Hz, pyH), 2.31 (3H, s, CH3). 13C{1H} NMR [CH3CN-d3, 125.7
MHz]: δ (ppm) 198.8 (C, CO) 198.7 (C, CO), 196.7 (C, CO), 153.0
(C, picCan), 152.1 (2C, picCHan), 149.8 (C, ArC-NO2), 149.2 (C,
pyCH), 142.5 (C, NCH), 139.4 (C, PyCH), 137.4 (C, ArCH),
136.3 (C, ArC), 130.2 (C, ArCH), 127.0 (2C, picCHan), 126.2 (C,
ArCH), 124.5 (C, ArCH), 113.4 (C, pyCH), 112.7 (C, pyCH), 23.40
(C, CH3). IR: ν (cm−1) 2029 (fac-ν(CO)), 1909 (fac-ν(CO)),
1526 (ArNO2, νas(NO)2), 1347 (ArNO2, νsym(NO)2). HRMS
(ESI+) m/z calcd for C15H10N3O3BrRe: 547.9620. Found: 548.0091
{[M + H]+, 100%}. RP-HPLC (System A): RT (min) 14.1. Crystals
suitable for X-ray crystallography were grown by slow evaporation the
reaction mixture.
(0.04 g, 55%). H NMR [DMSO-d6, 500 MHz]: δ (ppm) 8.69 (2H,
AA′XX′, J = 9.5 Hz, ArH), 8.24 (2H, AA′XX′, J = 9.0 Hz ArH), 8.16
(1H, dd, J = 6.0, 0.5 Hz, pyH), 7.66 (1H, s, NCH), 7.52 (1H, t, J =
6.5 Hz, 1.6 Hz, pyH), 7.00 (1H, d, J = 8.5 Hz, pyH), 6.44 (1H, t, J =
6.5 Hz, pyH), 4.13−4.04 (2H, m, NH2CH2CH2CH2N(CH3)2), 2.60−
2.53 (2H, m, NH2CH2CH2CH2N(CH3)2), 2.11 (2H, m,
NH2CH2CH2CH2N(CH3)2), 1.95 (6H, s, N(CH3)2), 1.47 (2H,
quintet, J = 6.5 Hz, NH2CH2CH2CH2N(CH3)2). 13C{1H} NMR
[DMSO-d6, 125.7 MHz]: δ (ppm) 198.3 (2C, CO), 194.5 (C, CO),
168.9 (C, pyC), 148.4 (C, ArNO2), 145.3 (C, NCH), 139.8
(pyCH), 139.3 (C, pyCH), 138.1 (C, ArC), 130.9 (2C, ArCH), 123.4
(2C, ArCH), 113.9 (C, pyCH), 111.9 (C, pyCH), 57.6 (C,
NH2CH2CH2CH2N(CH3)2), 47.3 (C, NH2CH2CH2CH2N(CH3)2),
44.8 (2C, N(CH3)2), 27.9 (C, NH2CH2CH2CH2N(CH3)2). IR: ν
(cm−1) 2007 (fac-ν(CO)), 1885 (fac-ν(CO)), 1874 (fac-ν(C
O)), 1460 (ArNO2, νas(NO)2), 1319 (ArNO2, νsym(NO)2). ESI-
MS m/z calcd for C20H24N6O5Re: 615.1366. Found: 615.1356 {[M +
H]+, 100%}. RP-HPLC (System A): RT (min) 10.7.
L5. To a solution of 1-(3-bromopropyl)-2-nitro-1H-imidazole37
(0.10 g, 0.43 mmol; 1) and di(2-picolyl)amine (0.08 mL, 0.43 mmol)
in anhydrous THF (3 mL) was added N,N-DIPEA (0.07 mL, 0.39
N
DOI: 10.1021/acs.inorgchem.5b01691
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
mmol). The mixture was heated at reflux for 2 h then cooled to RT.
The solution was reduced in vacuo and purified by column
chromatography (EtOAc/CH3OH, 60:40) to afford the desired
compound in a moderate yield as yellow-orange oil (0.08 g, 55%).
1H NMR [DMSO-d6, 500 MHz]: δ (ppm) 8.48 (2H, d, J = 4.0 Hz,
pyH), 7.76 (2H, td, J = 7.6, 1.8 Hz, pyH), 7.54 (1H, d, J = 1.0 Hz,
ImCH), 7.49 (2H, d, J = 7.8 Hz, pyH), 7.25 (2H, ddd, J = 7.4, 4.9, 1.1
Hz, pyH), 7.10 (1H, d, J = 1.0 Hz, ImCH), 4.38 (2H, t, J = 7.3 Hz,
CH2), 3.74 (4H, s, CH2), 2.02 (2H, quin, J = 7.2 Hz, CH2), 1.25 (2H,
m, CH2). 13C{1H} NMR [CH3CN-d6, 125.7 MHz]: δ (ppm) 158.9
(2C, pyC), 148.7 (2C, pyCH), 136.4 (2C, pyCH), 127.7 (C, CH),
127.6 (C, CH), 122.8 (2C, pyCH), 122.1 (2C, pyCH), 59.4 (2C,
CH2), 50.1 (C, CH2), 47.6 (C, CH2), 27.2 (C, CH2). IR: ν (cm−1)
1400 (ImNO2, νas(NO)2), 1321 (ImNO2, νsym(NO)2). HRMS
(ESI+) m/z calcd for C18H21N6O2: 353.1726. Found: 353.1723 {[M +
H]+, 100%}. RP-HPLC (System A): RT (min) 7.24.
(1H, t, J = 7.5 Hz, pyH), 7.69 (1H, s, ImCH), 7.67 (1H, d, pyH), 7.57
(1H, t, J = 6.0 Hz, pyCH), 7.23 (1H, s, ImCH), 4.77 (1H, d, 2JHH = 16
Hz, NCH2CO2), 4.51 (1H, obscd d, NCH2CO2), 4.49 (2H, br t,
2
NCH2CH2CH2Im), 3.82 (1H, d, JHH = 17 Hz, NCH2Py), 3.62−3.54
(2H, m, NCH2CH2CH2Im), 3.38 (1H, obsc’d d, NCH2Py), 2.39−2.28
(2H, m, NCH2CH2CH2Im). 13C{1H} NMR [DMSO-d6, 125.7 MHz]:
δ (ppm) 198.9 (C, CO), 196.5 (2C, CO), 185.6 (C, CO2), 161.0 (C,
pyC), 150.1 (C, pyCH), 146.4 (C, pyCH), 135.9 (C, ImCH), 130.1
(C, ImCH), 128.6 (C, pyCH), 127.6 (C, pyCH), 62.8 (C, CH2), 62.4
(C, CH2), 56.8 (C, CH2), 49.9 (C, CH2), 29.2 (C, CH2). IR: ν (cm−1)
2012 (fac-ν(CO)), 1900 (fac-ν(CO)), 1874 (fac-ν(CO)),
...
...
1652 (νas(C − O)2), 1485 (νas(NO)2), 1365 (νsym(C − O)2), 1339
(νsym(NO)2). ESI-MS m/z calcd for C17H17N5O7Re 590.0686.
Found: 590.0700 {[M + H]+, 100%}. RP-HPLC (System A): RT
(min) 10.8.
fac-[99mTc(CO)3(L5/6)]. Technetium-99m labeling of L5 and HL6
involved the initial synthesis of technetium tricarbonyl precursor, fac-
fac-[Re(CO)3(L5)]BPh4. A degassed solution containing [Re-
(H2O)5(OTf)] (0.09 g, 0.19 mmol) and 3-(2-nitro-1H-imidazol-1-
yl)-N,N-bis(2-pyridylmethyl)-1-propanamine (L5; 0.07 g, 0.19 mmol)
in methanol (5 mL) was subjected to microwave irradiation for 15 min
at 120 °C. The solution was reduced in vacuo, and a concentrated
solution of sodium tetraphenylborate (0.5 g, 1.4 mmol) in CH3OH (1
mL) was added. An off-white solid precipitated out of solution and was
collected via vacuum filtration (0.13 g, 73%). Crystals of X-ray
crystallographic quality were grown via slow evaporation of the solid
[
99mTc(H2O)3(CO)3]+. Sodium pertechnetate, Na[99mTcO4], was
99
eluted from a Gentech
Mo/99mTc sterile generator (1000 MBq,
Austin Health, Heidelberg, Australia, via ANSTO Health) as a 1.0 mL
saline solution (0.9% v/v). The radioactive solution was then added to
an evacuated Isolink vial (Mallinckrodt Pharmaceuticals, The Nether-
lands) and heated for 20 min at 100 °C. The solution was cooled to
room temperature. Separate solutions of ligands L5 and L6Et in
acetonitrile/water (1:2 v/v) were prepared (6.7 mg mL−1). To the
aqueous fac-[99mTc(H2O)3(CO)3]+ solution (0.1 mL) was added 0.1
M HCl, (∼25 μL), 0.1 M PBS buffer (50 μL pH 7.4), and the ligand
stock solution (20 μL). The pH was adjusted to 7−8 with additional
0.1 M HCl if required. The mixtures were heated to 65 °C for 30 min.
Aliquots (20 μL) of the reaction mixture were injected onto the
analytical RP-HPLC column (Column 7, System C) for UV and
radioactivity analysis.
Partition Coefficient and Stability Measurements for Radio-
labeled Complex fac-[99mTc(CO)3(L5)]+. Partition coefficients were
determined by the addition of fac-[99mTc(CO)3(L5)]+ (10 μL, ∼ 5
MBq without decay correction) to an octanol/0.1 M PBS (pH 7.4)
solution (1:1 v/v). The mixture was shaken for 3 min; then the layers
were left to separate. An aliquot of the organic layer (0.9 mL octanol)
was added to freshly prepared 0.1 M PBS (0.9 mL), and each phase
was presaturated with the other phase and shaken for 3 min. The
mixture was centrifuged (5 min, 13 200 rpm) and aliquots (0.5 mL)
from the two separate layers (aqueous and organic) taken for analysis
of the radioactive decay. All samples were prepared and measured in
triplicate.
For serum stability studies, 20 mL of blood from a healthy male was
centrifuged (10 min, 3000 rpm) to separate blood plasma and red
blood cells. The plasma was transferred to a separate vial and the red
blood cells discarded. An aliquot of plasma (0.6 mL) was added to
labeled compound fac-[99mTc(CO)3(L5)]+ (0.15 mL), the radioactivity
monitored, and the mixture then incubated at 37 °C. Aliquots (0.1
mL) of the mixture were removed from heating at 10 min and every 2
h following (over 6 h), and acetonitrile (0.1 mL) was added to the
serum/tracer mix to precipitate serum proteins. The suspension was
shaken for 5 min, then centrifuged (5 min, 13 200 rpm). The
radioactivity of the supernatant and pellet was recorded. The
supernatant (20 μL) was analyzed by analytical RP-HPLC (System
C) for UV and radioactivity analysis and the pellet washed with
acetonitrile (3 × 0.1 mL), and the radioactivity levels were again
recorded (radioactivity levels of the pellet were negligible).
1
dissolved in acetonitrile. H NMR [CH3CN-d3, 500 MHz]: δ (ppm)
8.75 (2H, s, pyH), 7.84 (2H, td, J = 7.8, 1.5 Hz, pyH), 7.39−7.36 (3H,
m, CH), 7.27 (8H, m, BPh4), 7.16 (1H, d, J = 1.0 Hz, CH), 6.99 (8H,
t, J = 7.8 Hz, BPh4), 6.84 (4H, t, J = 7.2 Hz, BPh4), 4.60 (4H, s, CH2),
4.51 (2H, t, J = 7.0 Hz, CH2), 3.86−3.83 (2H, m, CH2), 2.41−2.40
(2H, m, CH2). 13C{1H} NMR [CH3CN-d6, 125.7 MHz]: δ (ppm)
196.9 (C, CO), 196.8 (2C, CO), 165.4 (C, BPh4), 165.0 (C, BPh4),
164.6 (C, BPh4), 164.2 (C, BPh4), 161.1 (C, pyC), 153.1 (2C, pyCH),
141.4 (2C, pyCH), 136.7 (8C, BPh4), 129.2 (C, CH), 128.1 (C, CH),
126.7 (2C, pyCH), 126.6−126.5 (8C, BPh4), 124.5 (2C, pyCH), 122.8
(4C, BPh4), 68.4 (2C, CH2), 67.8 (C, CH2), 47.9 (C, CH2), 26.7 (C,
CH2). IR: ν (cm−1) 2027 (fac-ν(CO)), 1924 (fac-ν(CO)),1909
(fac-ν(CO)), 1482 (ImNO2, νas(NO)2), 1365 (ImNO2,
νsym(NO)2). HRMS (ESI+) m/z calcd for C20H15N5O5Re:
623.1053. Found: 623.1040 {[M]+, 100%}. RP-HPLC (System A):
RT (min) 4.97
L6Et. Ethyl N-(2-pyridylmethyl)amino acetate (0.09 g, 0.43 mmol;
2) was added to a solution of 1-(3-bromopropyl)-2-nitro-1H-imidazole
(0.10 g, 0.43 mmol; 1) in dry CH3CN (7 mL) and N,N-DIPEA (0.10
mL, 0.6 mmol). The reaction was heated to reflux overnight, then
cooled and concentrated in vacuo. Purification by column chromatog-
raphy (EtOAc/hexane, 96:4) afforded clear oil (0.03 g, 21%). 1H
NMR [CHCl3-d, 500 MHz]: δ (ppm) 8.87 (1H, ddd, J = 5.6, 1.6, 0.7
Hz, pyH), 8.26 (1H, td, J = 7.8, 1.7 Hz, pyH), 7.92 (1H, dd, J = 8.0, 0.5
Hz, pyH), 7.73 (1H, ddd, J = 7.6, 5.7, 1.3 Hz, pyH), 7.22 (1H, d, J =
1.1 Hz, ImCH), 7.12 (1H, d, J = 1.1 Hz, ImCH), 4.49−4.46 (2H, m,
CH2), 4.32 (2H, s, CH2), 4.19 (2H, q, J = 7.2 Hz, CH2), 3.54 (2H, s,
CH2), 2.90 (2H, t, J = 6.8 Hz, CH2), 2.06 (2H, quin, J = 7.3 Hz, CH2),
1.27 (3H, t, J = 7.2 Hz, CH2). 13C{1H} NMR [CHCl3-d, 125.7 MHz]:
δ (ppm) 170.2 (C, CO2Et), 155.2 (C, pyC), 144.0 (C, pyCH), 143.7
(C, pyCH), 128.6 (C, CH), 126.6 (C, CH), 126.3 (C, pyCH), 125.3
(C, pyCH), 61.4 (C, CH2), 56.0 (C, CH2), 54.4 (C, CH2), 51.4 (C,
CH2), 48.0 (C, CH2), 28.3 (C, CH2), 14.3 (C, CH3). IR: ν (cm−1)
1730 (m/sh, CO), 1555 (νas(NO)2), 1340 (νsym(NO)2).
HRMS (ESI+) m/z calcd for C16H22N5O4: 348.1672. Found: 348.1874
{[M + H]+, 100%}. RP-HPLC (System B): RT (min) 11.2.
Confocal Fluorescent Microscopy. Human carcinoma HeLa
cells were grown on poly-L-lysine (PLL) coated coverslips and
incubated at 37 °C (95% air, 5% CO2). When cells had reached
approximately 80% confluency, they were treated with 5, 10, 25, and
fac-[Re(CO)3(L6)]. Sodium hydroxide (3.45 mg, 0.09 mmol) and
L6Et (0.03 g, 0.09 mmol) in CH3CN (4 mL) were heated at reflux for
2 h. The solvent was reduced in vacuo and a solution of
[Re(CO)5OTf] (0.04 g, 0.09 mmol) in CH3OH (4 mL) added and
subjected to microwave irradiation for 15 min at 120 °C. The solvent
was concentrated to 1 mL and Et2O added. The precipitate was
collected via vacuum filtration and washed with copious amounts of
cold Et2O to afford an off-white solid (0.03 g, 60%). 1H NMR
[DMSO-d6, 500 MHz]: δ (ppm) 8.75 (1H, d, J = 4.5, Hz, pyH), 8.14
50 μM solutions of fac-[Re(CO)3(L1−2)(4-picoline)] in DMSO, λex
=
520 nm. Cell treatments were for 30 min. Cells were then rinsed with
1% PBS and fixed with 4% (w/v) paraformaldehyde for 20 min at
ambient temperature. Following fixation, cells were rinsed twice with
1% PBS and mounted onto glass slides using DAKO fluorescence
mounting medium, and images were collected. Images of the cells were
collected using an Olympus FV1000 IV81 confocal microscope.
O
DOI: 10.1021/acs.inorgchem.5b01691
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Cellular Assays. Cell uptake studies were carried out with the
assistance of Dr. Christine Schieber and Ms. Irene Volitakis.
Compounds were prepared as 10 mM stock solutions in culture-
grade DMSO, and the control compound [Re(CO)5OTf] was
prepared as a 10 mM stock solution in Milli-Q water. To each
complex sample (50 μL) was added warmed, serum-free media
(DMEM/F12 + P/S + NEAA + HEPES, 50 mL) for a final media
solution concentration of 10 μM.
SH-(SY5Y) cells were grown continuously and passaged at regular
intervals. Cells were removed from the tissue culture flask once 90%
confluent and then grown in Petri dishes until approximately 60%
confluency had been reached. Half the Petri dishes were placed in a
BD GasPak EZ gas generating chamber with two activated gas-
generating sachets (BD GasPak EZ ref: 260001, Anaerobe Container
System with Indicator) for 16 h. Low oxygen was confirmed for each
assay as per the manufacturer’s instructions.
For nonhypoxic (normoxic) cells, media were aspirated off the cells
that were not in the anaerobic chamber and replaced with 10 mL of
the media/complex solution (per plate in quadruplets). The cells were
then incubated for 60 min at 37 °C; DMSO was used as a vehicle
control whereby cells were treated and incubated under the same
conditions. For oxygen-depleted (hypoxic) cells, Petri dishes were
removed from the anaerobic container system, and the existing media
were replaced with fresh media solutions, which had been
deoxygenated with N2 gas over 10 min. Freshly prepared treatment
solution (10 μM media/complex) was also degassed and then added
to the cells after removal of the media. Cells were again placed in the
GasPak EZ container with three new GasPak bags and incubated for
60 min at 37 °C; DMSO was used as a vehicle control whereby cells
were treated and incubated under the same conditions.
Cells were scraped into the media and transferred into 15 mL
Eppendorf tubes, which were centrifuged for 3 min (1500 rpm). The
supernatant was removed and the pellet washed with PBS (2 × 5 mL).
The cell pellet was resuspended in 1 mL of PBS and 100 μL removed
for a BCA protein assay. The remaining mixture was centrifuged for 3
min (1500 rpm) and the PBS supernatant discarded. Cell pellets were
stored at −20 °C.
produced at Austin Health following a previously reported
procedure.57
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.5b01691.
■
S
Summary of crystal data and structure refinement for fac-
[Re(CO)3(HL3)(Br)] and fac-[Re(CO)3(HL4)(Br)] is
also given (PDF)
Crystallographic Information Files (CIF) for com-
pounds: HL1, HL2, fac-[Re(CO)3(HL1)Br], fac-[Re-
(CO)3(HL2)Br], fac-[Re(CO)3(HL3)Br], fac-[Re-
(CO)3(HL4)Br], fac-[Re(CO)3(HL1)(pyridine)]BPh4,
fac-[Re(CO)3(L1)(4-picoline)], fac-[Re(CO)3(L2)-
(pyridine)], fac-[Re(CO)3(L2)(4-picoline)], and fac-[Re-
(CO)3(L5)]BPh4. Cambridge Crystallographic Data
Centre numbers 1416475-1416485. Crystallographic
Information File (CIF)
Crystallographic Information File (CIF)
Crystallographic Information File (CIF)
Crystallographic Information File (CIF)
Crystallographic Information File (CIF)
Crystallographic Information File
(CIF)
Crystallographic Information File (CIF)
Crystallographic Information File (CIF)
Crystallographic Information File (CIF)
Crystallographic Information File (CIF)
Crystallographic Information File (CIF)
AUTHOR INFORMATION
Corresponding Author
*E-mail: pauld@unimelb.edu.au.
■
Rhenium levels were measured using a Varian UltraMass ICP-MS
instrument and quoted relative to protein measured by bicinchoninic
acid (BCA) protein assay. BCA protein assay was undertaken via a 10-
fold dilution (with PBS) of the 100 μL cell-pellet/PBS mixture
acquired earlier. A solution of BCA−reagent mixture, consisting of
reagent A and reagent B (50:1), was prepared. Working solutions and
standards (25 μL) were pipetted into 96-well microplates, followed by
the addition of BCA−reagent (200 μL per well). The plates were
covered in foil and incubated at 25 °C for 30 min, and the absorbance
was read at λ 562 nm for the colorimetric detection and quantitation of
total protein in the cells.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Animal SPECT Imaging Studies with fac-[99mTc(CO)3(L5)]+. All
animal experiments were approved by the Austin Hospital animal
ethics committee. SPECT/CT studies involved the same labeling
procedure as covered above, except that half the amount of
radioactivity was used (approximately 50 MBq) per sample. SK-RC-
52 tumors were first grown by injecting suspensions of 6 × 106 of SK-
RC-52 tumor cells in saline subcutaneously into the flank of the three
BALB/c nude mice. Tumours were allowed to grow to a size of about
300 mm3, then cut into 40 mm3 pieces and transplanted into the
shoulder of the BALB/c nude mice. Imaging was undertaken when the
tumors were 400 mm3 (Mouse 1), 600 mm3 (Mouse 2), and 1100
mm3 (Mouse 3) in size. A cannula was inserted into the lateral tail vein
of Mouse 1, Mouse 2, and Mouse 3 and 21, 18, and 19 MBq of fac-
■
The Australian Research Council and the National Health and
Medical Research Council are thanked for partial funding of
this research. The imaging equipment used for the animal
nanoSPECT/CT studies was purchased by the Australian
Cancer Research Foundation’s Centre for Translational Cancer
Therapeutics. P.S.D. is an ARC Future Fellow. A.J.N.
acknowledges the Albert Shimmins’ Postgraduate Writing-Up
Award.
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