Rhenium and Technetium-oxo Complexes with Thioamide Derivatives of Pyridylhydrazine Bifunctional Chelators Conjugated to the Tumour Targeting Peptides Octreotate and Cyclic-RGDfK

Article abstract of DOI:10.1021/acs.inorgchem.7b01247

This research aimed to develop new tumor targeted theranostic agents taking advantage of the similarities in coordination chemistry between technetium and rhenium. A γ-emitting radioactive isotope of technetium is commonly used in diagnostic imaging, and there are two β^- emitting radioactive isotopes of rhenium that have the potential to be of use in radiotherapy. Variants of the 6-hydrazinonicotinamide (HYNIC) bifunctional ligands have been prepared by appending thioamide functional groups to 6-hydrazinonicotinamide to form pyridylthiosemicarbazide ligands (SHYNIC). The new bidentate ligands were conjugated to the tumor targeting peptides Tyr³-octreotate and cyclic-RGD. The new ligands and conjugates were used to prepare well-defined {M=O}³⁺ complexes (where M = ^99mTc or ^natRe or ¹⁸⁸Re) that feature two targeting peptides attached to the single metal ion. These new SHYNIC ligands are capable of forming well-defined rhenium and technetium complexes and offer the possibility of using the ^99mTc imaging and ^188/186Re therapeutic matched pairs.

Full text of DOI:10.1021/acs.inorgchem.7b01247

Article
pubs.acs.org/IC
Rhenium and Technetium-oxo Complexes with Thioamide
Derivatives of Pyridylhydrazine Bifunctional Chelators Conjugated to
the Tumour Targeting Peptides Octreotate and Cyclic-RGDfK
†
†
‡
‡
§
Andrea J. North, John A. Karas, Michelle T. Ma, Philip J. Blower, Uwe Ackermann,
Jonathan M. White,† and Paul S. Donnelly*,†
†The School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, 3010, Victorria,
Australia
‡
Division of Imaging Sciences and Biomedical Engineering, King’s College London, Fourth Floor Lambeth Wing, St Thomas’
Hospital, London SE1 7EH, U.K.
§
Department of Molecular Imaging and Therapy, Department of Medicine, University of Melbourne, Austin Health, Studley Road,
Heidelberg, Victoria 3010, Australia
*
S Supporting Information
ABSTRACT: This research aimed to develop new tumor
targeted theranostic agents taking advantage of the similarities
in coordination chemistry between technetium and rhenium. A γ-
emitting radioactive isotope of technetium is commonly used in
−
diagnostic imaging, and there are two β emitting radioactive
isotopes of rhenium that have the potential to be of use in
radiotherapy. Variants of the 6-hydrazinonicotinamide (HYNIC)
bifunctional ligands have been prepared by appending thioamide
functional groups to 6-hydrazinonicotinamide to form pyridylth-
3
iosemicarbazide ligands (SHYNIC). The new bidentate ligands were conjugated to the tumor targeting peptides Tyr -octreotate
3+
99m
and cyclic-RGD. The new ligands and conjugates were used to prepare well-defined {MO} complexes (where M = Tc or
nat
188
Re or Re) that feature two targeting peptides attached to the single metal ion. These new SHYNIC ligands are capable of
99m
188/186
forming well-defined rhenium and technetium complexes and offer the possibility of using the
Tc imaging and
Re
therapeutic matched pairs.
and therapeutic (186/188Re) matched pair using a single targeted
ligand to form essentially isostructural complexes.
INTRODUCTION
■
2
−6
The technetium-99m isotope has excellent properties for
detection with single photon emission computed tomography
SPECT) due to its low energy and nonparticulate gamma-ray
emission (t1/2 = 6.01 h, Emax = 141 keV γ-ray emission, λ < 10
pm). Despite recent concerns over production related shortages
of technetium-99m and the advent of positron emission
tomography technetium-99m retains its importance to nuclear
medicine to the extent that the isotope is used in over 80% of
One approach to targeted imaging and therapy is to
incorporate appropriate metal radionuclides into coordination
complexes that are attached to biological targeting vectors such
as tumor targeting peptides, antibodies or antibody fragments.
Peptides that feature the −RGD- (arginine-glycine-aspartic
acid) fibronectin fragment such as the cyclic-RGDfK
pentapeptide (cRGDfK) bind to α β integrin receptors that
(
v
3
are overexpressed in certain invasive tumors including
1
nuclear imaging procedures worldwide. The heavier third row
osteosarcomas, glioblastoma, melanomas, and breast cancer,
7
−16
Group VII congener, rhenium, has an ionic radius similar to
technetium due to the lanthanide contraction. Technetium and
rhenium display similar coordination chemistry often resulting
in essentially isostructural technetium and rhenium complexes.
It is common for technetium and rhenium complexes to be
essentially isostructural. There are two isotopes of rhenium that
and can be used to selectively target tumor cells.
Metabolically stabilized somatostatin analogues such as
octreotide and octreotate bind to somatostatin subtype 2
receptors (sstr2) that are overexpressed in many types of
neuroendocrine tumors compared to relatively low levels of
17−21
expression in other tissues and organs.
are of potential use in targeted radiotherapeutics, rhenium-186
Tumor targeting technetium based imaging agents can be
prepared using 6-hydrazinonicotinamide (HYNIC) derivatives
conjugated to targeting molecules as ligands to form
−
(t1/2 = 89.3 h, Emax = 1.07 MeV β particle emission, 137 keV γ-
ray emission) and rhenium-188 ((t = 16.9 h, Emax = 2.12
1
/2
−
MeV β particle emission, 155 keV γ-ray emission). The similar
coordination chemistry of technetium and rhenium offers the
possibility of using their radioisotopes as an imaging (9 Tc)
Received: May 17, 2017
9m
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.7b01247
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Inorganic Chemistry
Article
technetium(III) diazenido complexes. 2−31 The HYNIC ligand
forms remarkably stable technetium complexes, and manipu-
lation of the carboxylate functional group to attach a variety of
targeting molecules is generally straightforward. HYNIC binds
to technetium through the terminal hydrazine nitrogen but
probably forms bidentate complexes through coordination to
2
Modification of the terminal hydrazinic nitrogen of
hydrazinopyridine to incorporate an additional thiourea
functional group results in a ligand system that is capable of
V
3+
forming well-defined, very stable complexes with {Re O}
cores while retaining the bioconjugation possibilities well
4
4,45
established for HYNIC.
A preliminary communication
2
9,32
V
the pyridyl nitrogen.
In all the crystallographically
characterized technetium and rhenium complexes with one or
more HYNIC-like ligands, such as 2-hydrazinopyridine, the
reported the structural characterization of a Re -oxo complex
featuring two pyridylphenylthiocarbazide (SHYNIC) ligands
44
(Figure 1). In this manuscript we extend this concept by
synthesizing a family of different substituted pyridylthiosemi-
carbazide ligands with carboxylate or ester functional groups
that were used to tether octreotate and cyclic-RGD peptides to
33
pyridyl nitrogen is also coordinated to the metal center.
A
variety of coligands such as tricine, nicotinic acid, EDDA, and
phosphines (TPPTS, TPPDS, TPPMS = tri/bi/sodium
triphenylphosphine tri/di/monosulfonate) are required to
complete the coordination sphere and stabilize the metal
oxidation state, and this leads to a high degree of uncertainty in
the exact nature of the primary coordination sphere as well as
challenges in ensuring structural homogeneity in the formulated
product. Variation of the coligand can modify the in vivo
metabolism and excretion. A well-established “ternary ligand
system” involves combining the HYNIC ligand with
tetradentate tricine and monodentate trisodium 3,3′,3″-
phosphanetriyltris(benzenesulfonate) (TPPTS) coligands, but
the possibility of forming multiple isomers adds complica-
3
+
the ligands. The new ligands were used to prepare {MO}
complexes (where M = Tc or Re) that feature two targeting
peptides attached to the single metal ion. These modified
HYNIC ligands are capable of forming well-defined rhenium
and technetium complexes and offer the possibility of using the
two radionuclides as imaging and therapeutic matched pairs.
34
RESULTS AND DISCUSSION
■
1
−4
Synthesis of H L , and Their Ester Derivatives,
2
1
−4
3+
H L (OMe) and {ReO} Complexes. Synthesis of 6-
2
hydrazinonictonic acid (HYNIC), 2, required treatment of 6-
3
3,35−39
47
tions.
chloronicotinic acid (1) with aqueous hydrazine. Ligands
1
4
Despite the superficial similarities in coordination chemistry
between technetium and rhenium extrapolation of the HYNIC
strategy to radioactive rhenium isotopes is challenging
presumably due to their differences in kinetic lability and
redox chemistry. Some of the difficulty in isolating pure Re-
HYNIC-peptide conjugates can be understood by considering
H L to H L were prepared by reaction of either the ethyl, tert-
2 2
butyl, phenyl, or nitrophenyl isothiocyanate with 6-hydrazino-
nictonic acid (1) in anhydrous N,N-dimethylacetamide (DMA)
(Scheme 1).
3
3
The rhenium complexes of the methyl ester derivatives of
1−3
1−3
+
H2L
complexes, [ReO(HL (OMe)) ] , can be prepared
2
−
3,40
the reaction of [ReO ] with 2-hydrazinopyridine (used as
by reaction of the either trans-[ReOCl (PPh ) ] or [tBu N]-
4
3
3 2
4
3
model for HYNIC).
This reaction results in relatively
[ReOCl ] with the two equivalents of ligand (Scheme 2). The
4
1
−3
+
complex coordination chemistry due to the ability of the
pyridylhydrazine derived ligands to coordinate as either
monodentate or bidentate ligands and the existence of protic
equilibria as well as the formation of complexes where two
pyridylhydrazine derived units are coordinated to the rhenium
IR spectra for the three complexes, [ReO(H2L (OMe)) ] ,
2
−
1
display medium intensity bands at ν 960−963 cm character-
̅
48
istic of ReO stretches. Bands, which occur between ν 1553
̅
and 1557 cm− due to carbonyl stretching of the ester
1
−1
functional group, shift approximately 150 cm lower in energy
when compared to the metal-free ligands.
Analysis of the complexes by H NMR data reveals that the
2
6,33,40−43
(Figure 1).
1
two coordinated ligands are magnetically equivalent, with three
resonances at δ 8.63, 8.25, and 7.86 ppm corresponding to the
1
+
six pyridinyl CH protons for [ReO(HL (OMe)) ] , and similar
2
resonances for the phenyl and tert-butyl derivatives. The
pyridine proton which is closest to the rhenium ion shifts from
δ 6.55 ppm in free ligand to δ 7.86 ppm in the complex. The
methyl ester functional group gives rise to singlets at δ 3.89
(
DMSO-d ), 3.86 (CHCl -d), and 3.92 (DMSO-d ) for
6 3 6
1
,2,3
+
complexes [ReO((HL )(OMe)) ] respectively. The [ReO-
2
1
+
(
HL (OMe)) ] complex was stable to cysteine and histidine
2
challenge experiments with very little decomposition evident
<5%), as detected by analysis by HPLC and UV/vis
(
spectroscopy, when incubated at 37 °C for 24 h in the
presence of a 100-fold excess of cysteine and histidine.
1
Red crystals of [ReO(HL (OMe)) ]CF CO suitable for X-
2
3
2
ray crystallographic analysis were obtained by evaporation of a
solution of the compound that had been purified by
semipreparative HPLC using an aqueous/CH CN mobile
phase with 0.1% trifluoracetic acid (Figure 2a). The compound
3
Figure 1. (a) 6-Hydrazinonicotinic acid. (b) Metal complex (M = Tc
or Re) with 6-hydrazinonicotinimide (HYNIC) acting as a
monodentate ligand. It is necessary to complete the coordination
sphere with coligands (L). (c) Metal complex (M = Tc or Re) with 6-
hydrazinonicotinimide (HYNIC) acting as a bidentate ligand. (d)
̅
crystallizes in the triclinic space group, P1, and the rhenium ion
is in a distorted square pyramidal environment with the oxo
group in the apical position relative to the pseudo basal plane of
two five-membered chelate rings. Each thiocarbahydrazide
functional group is doubly deprotonated and serves as a
V
Pyridylphenylthiocarbazide (SHYNIC) ligand. (e) Re -oxo complex
44
featuring two SHYNIC ligands.
B
DOI: 10.1021/acs.inorgchem.7b01247
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Inorganic Chemistry
Scheme 1. Synthesis of Ligands H L −H L and Their Methyl Ester Derivatives H L (OMe)−H L (OMe)
Article
1
4
1
4
2
2
2
2
Scheme 2. Synthesis of Rhenium Complexes
N -Fmoc-L-Lys, was immobilized on chlorotrityl resin, and the
ε
1
‑3
+
[
ReO(HL )(OMe)) ]
N -Fmoc group was removed by treatment with piperidine.
ε
2
1
The ligand, H L , was added to the resin in a mixture of DMF
2
followed by the coupling agent HATU (HATU = 1-
[
bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]-
pyridinium-3-oxid hexafluorophosphate) in the presence of
N,N-diisopropylethylamine (DIPEA). The product was cleaved
from the resin using trifluoroacetic acid that also resulted in the
deprotection of the N -t-butoxycarbonyl group (Scheme 3).
α
This lysine amino acid conjugate provides an amino acid with
an appended chelator, which with appropriate protecting
groups, could be incorporated into biological targeting
molecules with total site specificity via solid-phase peptide
59−63
synthesis.
The rhenium complex, [ReO(HL (Lys)) ] , was prepared by
1
+
2
adding either trans-[ReOCl (PPh ) ] or [tBuN][ReOCl ]
dianionic ligand fragment and a N,N/S,S trans configuration
about the Re-oxo. The selective formation of the N,N/S,S trans
geometric isomer presumably reflects a strong “trans effect”,
3
3 2
4
suspended in DMF to the reaction mixture, while the ligand
remained immobilized on the resin. Performing the complex-
ation while the ligand remained immobilized on the resin and
with the amino group still protected ensured that the functional
groups of the lysine did not complicate the coordination
49−51
although steric requirements may also play some role.
Protonation of the pyridyl nitrogen atom in each ligand results
in each ligand having a single negative charge and resulting an
overall monocationic complex. The two pyridinium protons
and the hydrogen atoms of the ethylamino functional group are
involved in hydrogen bonds interactions leading to a hydrogen
bonded centrosymmetric dimer (Figure 2b) with the two
remaining H-bond donors capped by water molecules.
chemistry. When green trans-[ReOCl (PPh ) ] is used as the
3
3 2
starting material the green colored suspension gradually
changes to colorless, and the resin beads turn dark red
1
+
indicative of the formation of [ReO(HL (Lys)) ] . After being
2
stirred at room temperature, the resin was washed with
dimethylformamide and dichloromethane and cleaved off the
resin with a 10% trifluoroacetic acid/dichloromethane mixture
The Re−O1 bond distance (1.679(3) Å) is typical for five-
coordinate, rhenium(V)-monooxo complexes and consistent
−
1
52−55
1
+
(
Scheme 3). Analysis of [ReO(HL (Lys)) ] complex by
with IR spectroscopy (ReO, ν 963 cm ).
The N2−C6
2
̅
and N6−C14 bond lengths (1.287(6) Å) are significantly
shorter than the other bonds within the chelate ring suggesting
electrospray ionization mass spectrometry (ESI-MS) reveals the
1
expected peaks. Analysis by H NMR shows the singlet
2
2
significant sp hybridized CN bond character. The Re−N
attributed to the aromatic proton on the pyridine ring (pyH )
bond distances average 2.05 Å and are marginally shorter than
shifts upon coordination to the metal center from δ 8.48 in
1
1
+
typical Re−N bonds (ca. 2.15−2.18 Å) suggesting some degree
H
2
L (Lys) to 8.58 ppm in [ReO(HL (Lys))
2
] . The four
5
1,56
13
1
1
+
of multiple bond character.
The Re−N1−N2 and Re−N5−
downfield C{ H} NMR signals in [ReO(HL (Lys))2] , 173.0
2
6
N6 bond angles average 124°, suggesting an approximately sp
hybridized nitrogen. The Re−S bond distances average 2.29 Å
and are similar to the Re−S bond distances in rhenium(V)
complexes with amino thiolate ligands and Re−S bond
distances in rhenium complexes with thiosemicarbazonato
(CO
2
H), 169.0 (NCS), 165.4 (CONH), and 163.6 (pyC ),
were assigned using HSQC and HMBC techniques.
Synthesis of Peptide-Conjugated Ligands,
1
−3
1−3
3
H
L
(cRGDfK) and H
L
(Tyr -Octreotate), and Corre-
2
2
1
−3
sponding Rhenium Complexes [ReO((HL )(cRGDfK))
]
2
5
1,57,58
1−3
3
ligands.
The potential of the substituted pyridylthiosemicarbazide
and [ReO((HL )(Tyr -Octreotate)) ]. The cyclic pentapep-
2
tide, cRGDfK, was prepared using standard solid phase peptide
synthesis techniques with Fmoc (Fmoc = 9-fluorenylmethylox-
ycarbonyl) protected amino acids, using HATU/DIPEA
coupling methodology on chlorotrityl resin. The Fmoc
protecting groups were removed with 20% piperidine in
1
−4
(SHYNIC) ligands H2L
to be modified with amino acids
using standard solid phase peptide synthesis techniques was
1
first accomplished by attaching L-lysine to H2L to give
1
H L (Lys). The doubly N-protected lysine derivative, N -tBoc-
2
α
C
DOI: 10.1021/acs.inorgchem.7b01247
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
1
+
Figure 2. (a) ORTEP representation of [ReO(HL (OMe)) ] (50% probability ellipsoids). The trifluoroacetate counterion and hydrogen atoms
2
(
except those bound to nitrogen) are omitted. (b) Representation of hydrogen bonded centrosymmetric dimer with the two remaining H-bond
donors capped by water molecules.
1
a
Table 1. Selected Bond Lengths (Å) and Angles (deg) for the Rhenium Complex for [ReO(HL (OMe)) ]CF CO
2
2
3
Bond Lengths
Re−O(1)
N(1)−C(1)
C(6)−N(3)
C(14)−N(7)
1.679(3)
1.350(6)
1.343(6)
1.350(6)
1.356(6)
Re−S(1)
Re−N(1)
N(1)−N(2)
N(2)−C(6)
C(6)−S(1)
2.2820(11)
2.049(4)
1.422(5)
1.287(6)
1.784(5)
Re−S(2)
Re−N(5)
N(5)−N(6)
N(6)−C(14)
C(14)−S(2)
2.2917(11)
2.050(4)
1.418(5)
1.287(6)
1.769(4)
N(5)−C(9)
Bond Angles
O(1)−Re−S(1)
O(1)−Re−S(2)
O(1)−Re−N(1)
O(1)−Re−N(5)
N(1)−Re−N(5)
S(1)−Re−S(2)
115.44(11)
116.75(11)
101.73(15)
102.61(15)
155.64(15)
127.80(4)
N(1)−Re−S(1)
N(5)−Re−S(2)
N(5)−Re−S(1)
N(1)−Re−S(2)
C(1)−N(1)−Re
C(9)−N(5)−Re
80.74(11)
80.23(11)
89.71(11)
87.98(11)
125.0(3)
126.2(3)
Torsion Angles
N(4)−C(1)−N(1)−Re
N(8)−C(9)−N(5)−Re
N(4)−C(1)−N(1)−N(2)
N(8)−C(9)−N(5)−N(6)
C(2)−C(1)−N(1)−N(2)
C(10)−C(9)−N(5)−N(6)
169.5(3)
173.3(3)
−10.5(6)
−7.8(6)
167.1(4)
168.4(4)
N(6)−C(14)−N(7)−C(15)
N(2)−C(6)−N(3)−C(7)
N(2)−C(6)−S(1)−Re
N(6)−C(14)−S(2)−Re
N(3)−C(6)−S(1)−Re
N(7)−C(14)−S(2)−Re
13.8(7)
5.1(7)
3.7(4)
4.1(4)
−174.7(3)
−175.1(3)
a
Trifluoroacetate counterion bond lengths and angles are not provided.
1
DMF on the solid phase, followed by cleavage from the resin
reaction for the ethyl-SHYNIC derivative, H L (cRGDfK),
2
2
and cyclization using a small modification of published
resulted in a higher isolated yield (80%) than H L (cRGDfK)
2
6
4
1−3
3
procedures. The ligands, H2L , were conjugated to cRGDfK
using standard peptide coupling conditions (HATU, DIPEA)
to give H2L1 (cRGDfK) (Scheme 4). The new conjugates
were purified by semipreparative RP-HPLC and characterized
by electrospray mass spectrometry and analytical HPLC. The
(24%) and H L (cRGDfK) (55%). Analysis of aqueous
solutions of H2L (cRGDfK) by RP-HPLC revealed no
2
1
−3
−3
degradation over a 24 h.
The {ReO}3+ complexes of H L1−3(cRGDfK) were prepared
by adding [ReOCl ] in DMF at ambient temperature followed
2
−
4
D
DOI: 10.1021/acs.inorgchem.7b01247
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Inorganic Chemistry
Article
1
a
Table 2. Summary of Crystal Data and Structure Refinement for [ReO(HL (OMe)) ]CF CO
2
3
2
data collection
compound details
data collection
compound details
empirical formula
formula weight
C H N O ReS .H O.CF CO
2
V (Å3)
1551.05(13)
2
0
26
8
5
2
2
3
839.84
Z
2
3
(Mg m−3
)
crystal size (mm )
0.26 × 0.10 × 0.022
triclinic
D
1.797
calc.
crystal system
space group
T (K)
μ (mm−1)
9.599
P1
̅
F(000)
828
130.00(10)
reflections measured
10363
λ (Å)
1.54184
independent reflections
5568 [Rint = 0.0449]
a (Å)
10.9154(4)
11.1201(6)
13.2651(7)
86.355(4)
74.965(4)
87.273(4)
final R indices [I > 2σ(I)]
final R indices (all data)
goodness-of fit on F2
R = 0.0329
1
2
b (Å)
wR(F ) = 0.0794
c (Å)
R = 0.0398
1
2
α (deg)
β (deg)
γ (deg)
wR(F ) = 0.0831
1.027
a
Crystals were grown from a concentrated solution of the complex in methanol.
1
1
+
Scheme 3. “On-Resin” Formation of H L (Lys) and the Corresponding Rhenium-oxo Complex, [ReO(HL (Lys)) ]
2
2
by isolation and purification using semipreparative RP-HPLC
residues. The loss of H S results in the formation of a
2
(
2
Scheme 4). Analysis of the complexes by ESI-MS revealed the
carbodiimide form of the SHYNIC ligands. The formation of
65
+ molecular ion peaks at m/z 926.343, 954.372, and 974.371
carbodiimides from thioureas is well-known. This degradation
and loss of sulfur were not observed for RGD-based conjugates,
suggesting that in the case of the octreotate conjugates,
thiocarbazide-thiol-disulfide interchange/scrambling promotes
1
+
2
+
for [ReO(HL (cRGDfK)) ] , [ReO(HL (cRGDfK)) ] , and
2
2
3
+
[
ReO(HL (cRGDfK)) ] respectively.
2
An intramolecular disulfide bridge between the second and
3
seventh cysteine residues in Tyr -octreotate improves the
metabolic stability of the peptide, and this disulfide is often
introduced by oxidation of the linear octapeptide with 2,2′-
dithiodipyridine. Unfortunately the bioconjugation of ligands
the loss of H S from the ligands (Scheme 5).
2
As conventional off-resin oxidative cyclization methodologies
1
−3
3
were inadequate for this synthesis, H2L (Tyr -Oct) was
prepared entirely on solid support, where intramolecular
H2L1 to Tyr -octreotate was complicated by degradation of
−3
3
oxidation/cyclization preceded bioconjugation of H2L . The
1−3
1
−3
the pyridylthiocarbazide (SHYNIC) ligands (H2L ) in the
presence of the two cysteine thiol containing residues in the
linear peptide, leading to loss of H S identified in the ESI-MS
by a loss of 34 atomic mass units. This loss of H S from the
ligand was most prominent during reactions attempting
intramolecular oxidation of thiol groups in the two cysteine
eight-residue peptide was synthesized by sequential addition of
the amino acid residues via solid-phase peptide synthesis, using
acetamidomethyl (Acm) protected cysteine residues followed
by in situ Acm removal and simultaneous disulfide bond
2
2
66
formation using thallium(III) trifluoroacetate. Following
1
−3
cyclization, the preactivated SHYNIC derivative (H2L ) is
E
DOI: 10.1021/acs.inorgchem.7b01247
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Inorganic Chemistry
Scheme 4. Synthesis of H L (cRGDfK) and [ReO(HL (cRGDfK)) ]
Article
1
‑3
1‑3
+
2
2
Scheme 5. Suggested Mechanism for the Formation of a Carbodiimide from H2L1−3(Tyr -Oct) Resulting in Loss of H S
3
2
1
88
−
reacted at the deprotected D-phenylalanine N-terminus.
Cleavage and deprotection of remaining protecting groups
are achieved by treatment with trifluoracetic acid (Scheme 6).
performed using generator-produced [ ReO ] . A solution of
4
188
−
[
ReO ] , in an aqueous mixture of sodium chloride (0.9%
w/v concentration) and sodium tartrate, was reduced with
stannous chloride. This mixture was then reacted with
H L (Tyr -Oct) at 100 °C, leading to the formation of
[ ReO(HL (Tyr -Oct)) ] in ca. 67% radiochemical yield.
2
4
1−3
3
The rhenium complexes of H2L (Tyr -Oct) could be
1
3
prepared on-resin or in-solution by treatment with [tBu N]-
4
2
1
88
1
3
+
[
ReOCl ] in methanol (Scheme 6). The on-resin approach is
4
potentially of interest in producing radioactive complexes in
The compound was characterized by analytical reversed phased
−
188
1
3
+
high specific activity as unreacted [ReO ] and other impurities
HPLC (Figure 4), where [ ReO(HL (Tyr -Oct)) ] (reten-
4
2
such as colloidal rhenium could be readily removed by filtration
of the resin. The pure complex can be cleaved from the resin
with 50% trifluoracetic acid and is stable to this relatively high
concentration of acid. Analysis by HPLC and ESI-MS
confirmed the identity of the complexes, with the [ReO-
tion time = 11.6 min, detected using a NaI(Tl)) elutes with a
nat
similar retention time to the nonradioactive analogue [ ReO-
1
3
+
(HL (Tyr -Oct)) ] (retention time = 11.3 min, detected at
2
nat
λ220), where Re refers to naturally abundant Re isotopes. The
small difference in retention times is due to the different
configurations of the radioactivity and UV detectors. This
1
−3
3
+
(
HL (Tyr -Oct)) ] complexes showing signals in the ESI-
2
1
3
+
MS that could be attributed to the 3+ molecular ion with
expected rhenium isotope peak patterns (Figure 3).
elution profile of [ReO(HL (Tyr -Oct)) ] is distinct from that
2
1
3
of the free ligand, H L (Tyr -Oct) that elutes at 9.7 min under
2
1
88
1
3
+
188
Preparation of [ Re(HL (Tyr -Oct)) ] . Preliminary
the same conditions. Unreacted
Re species, presumably
2
1
3
188
188
−
radiolabeling of H L (Tyr -Oct) with radioactive Re was
[
ReO ] , elute with the solvent front at 2.1 min (Figure 4).
2
4
F
DOI: 10.1021/acs.inorgchem.7b01247
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
1‑3
3
Scheme 6. Reaction Scheme for the Formation of Octreotate-Derived Ligands H L (Tyr -Oct) and Corresponding Complexes,
2
1
‑3
+
[
ReO(HL (Tyr -Oct)) ]
3 2
3
1−3
3
+
Figure 3. RP-HPLC chromatogram (UV absorbance, a.u., λ 254 nm) of Tyr -octreotate rhenium complexes, [ReO(HL (Tyr -Oct)) ] . Inset:
2
Positive ion ESI-MS data of major isotope peaks corresponding to each complex as the tripositive cation, [ReO(HL1 (Tyr -Oct)) ] .
−3
3
3+
2
9
9m
99m
[99mTcO(HL1−3(Tyr -Oct)) ] were prepared in ca. 60−80%
3
+
Preparation of
Tc-Labeled Complexes, [ TcO-
2
1
−3
+
99m
1−3
3
+
(
HL (cRGDfK)) ] and [ TcO(HL (Tyr -Oct)) ] . The
technetium-99m complexes [ TcO(HL (cRGDfK)) ] and
radiochemical yield using mild conditions and relatively simple
procedures (Supporting Information, Figures S1 and S2).
2
2
9
9m
1−3
+
2
G
DOI: 10.1021/acs.inorgchem.7b01247
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
3
,63
challenging.
This family of bidentate ligands form stable
3+
complexes with the {ReO} core with two ligands coordinated
to a single metal ion. A rhenium complex with a methyl ester
functional group has been characterized by X-ray crystallog-
raphy and features the rhenium ion in a distorted square
pyramidal environment with the oxo group in the apical
position relative to the pseudo basal plane of two five-
membered chelate rings with a N,N/S,S trans configuration
about the Re-oxo core. The basic ligands have been decorated
3
with the tumor targeting peptides cyclic-RGD and Tyr -
octreotate, and these conjugates form complexes with rhenium
1
−3
to give well-defined single species, [ReO((HL )(cRGDfK))2]
1
−3
3
and [ReO((HL )(Tyr -octreotate)) ], without having to add
2
Figure 4. RP-HPLC chromatogram (UV absorbance, λ 224 nm and
coligands resulting in the formation of a single structural and
geometrical isomer. It is possible to form the rhenium
complexes using either standard solution chemistry or “on-
188/nat
1
3
+
radiation detection) of [
ReO(HL (Tyr -Oct)) ] and free ligand
2
1
3
H L (Tyr -Oct). The signal at 2.1 min in the chromatogram of
2
1
88
1
3
+
188
[
ReO(HL (Tyr -Oct)) ] corresponds to unreacted Re species.
2
resin”, and the latter approach may prove useful in isolating
radioactive 1
88/186
Re analogues in high specific activity. These
Preparation of [ TcO(HL (cRGDfK)) ]+ and [99mTcO-
9
9m
1−3
2
HL1 (Tyr -Oct)) ] involved adding an excess of the
−3
3
+
complexes feature two targeting peptides separated by 14
chemical bonds, and there is evidence that molecules
containing more than one targeting peptide, sometimes
referred to as bivalent, can display enhanced receptor binding
due to simultaneous binding to more than one receptor on the
(
2
appropriate ligand dissolved in aqueous sodium chloride (0.5
mg mL , 0.9% NaCl, pH 7.4) to a mixture of [ TcO ] that
−1
99m
−
4
has been reduced by tin(II) chloride in 0.1 M HCl in the
presence of tartrate (pH 1−4) at room temperature. The
1
4,67−72
radiolabeled 99mTc complexes were characterized by analysis by
surface on any given cell.
It is possible to prepare
188
1
3
+
[
ReO(HL (Tyr -Oct)) ] in ∼67% yield from generator
2
HPLC equipped with a radioactivity detector, and the elution
profiles were compared to the analogous nonradioactive
rhenium compounds (detected by UV absorbance). The close
correlation between the retention times of the rhenium and
technetium complexes strongly suggests that the complexes are
isostructural (Table 3). The small difference in the retention
188
−
produced [ ReO ] , and improved yields should be possible
4
by optimizing the reaction conditions. The analogous
1
−3
technetium complexes, [TcO((HL )(cRGDfK)) ] and
2
1
−3
3
[
TcO((HL )(Tyr -Octreotate)) ], were prepared directly
from [ TcO ] with tin chloride acting as a reducing agent.
2
99m
−
4
Comparison of HPLC profiles suggests the rhenium and
technetium complexes are isostructural. The complexes
described in this manuscript have two ligands coordinated to
a single metal ion, whereas conventional HYNIC systems
involve one HYNIC ligand binding to one metal ion. It is likely
the two different systems will exhibit quite different
biodistribution in vivo. These new systems warrant further
investigation as potential theranostic agents employing an
Table 3. RP-HPLC Retention Times (min) for Ligands and
+
1
2
[
MO(HL) ] Complexes (M = Re, 99 mTc)·L (ethyl), L (t-
2
3
a
butyl), and L (phenyl) Compounds
compound
H Lx
rhenium complex
technetium complex
2
1
L -cRGDfK
10.0
9.3
11.4
10.3
12.0
11.4
11.7
12.9
11.5
10.9
12.7
11.9
12.1
13.0
2
L -cRGDfK
imaging (9 Tc) and therapeutic (
9m
186/188
Re) matched pair for a
3
L -cRGDfK
10.9
10.1
10.9
11.0
1
3
single targeted agent.
L -(Tyr -Oct)
2
3
General Experimental. All reagents were purchased from
standard commercial sources. Nuclear magnetic resonance
(NMR) spectra were acquired on either an Agilent 400-MR
L -(Tyr -Oct)
3
3
L -(Tyr -Oct)
a
Linear gradient from 0 to 90% Buffer B to A. Buffer A: 0.1% TFA in
1
13
1
(
H NMR at 400 MHz and C{ H} NMR at 101 MHz) or a
Milli-Q water. Buffer B: 0.1% TFA in CH CN.
3
1
Varian FT-NMR 500 spectrometer ( H NMR at 500 MHz and
1
3
1
C{ H} NMR at 126 MHz) at 298 K. Chemical shifts were
times between the traces for 99mTc and Re complexes is, in part,
due to the detector configurations but could also reflect the
difference in polarity between the oxorhenium(V) and
oxotechnetium(V) cores.
referenced to residual solvent peaks and are quoted in ppm
relative to TMS.
Fmoc-L-amino acids, Fmoc-D-amino acids, Nvoc-Cl, HATU,
DIC, Wang resin, 2-chlorotrityl, Fmoc-Lys(ivDde)−OH, and
Fmoc-Cys(Acm)−OH were purchased from standard commer-
cial sources.
9
9m
3
3
+
The stability of [ TcO(HL (Tyr -Oct)) ] was assessed by
2
incubation in human plasma at 37 °C. The complex was stable
for at least 2 h with only small amounts of degradation products
Linear protected RGDfK peptide (Arg(Pbf)-Gly(tBoc)-
Asp(OtBu)-dPhe-Lys(tBoc)) was synthesized manually using
standard Fmoc solid phase peptide synthesis (SPPS)
procedures on the 2-chlorotrityl chloride resin. The linear
pentapeptide was cleaved from the resin (with retention of
protecting groups) using 1% TFA in dichloromethane and
shaking for 40 min. The mixture was filtered and the filtrate was
reduced in volume to afford crude linear product. Cyclization
involved reacting the crude material in a mixture of
(
<5%) evident that, based on their retention times in analytical
HPLC, are most likely due to degradation of the peptide.
CONCLUDING REMARKS
■
The new pyridylthiocarbazide ligands (SHYNIC, H L1−3)
2
described here offer a useful alternative to the standard
HYNIC system. While HYNIC has proved a very successful
9
9m
and versatile bifunctional ligand for
Tc coligands are
−1
required to complete the coordination sphere of the metal
ion and extrapolation to radioactive rhenium isotopes has been
dichloromethane (1 mg mL ), HATU (0.9 equiv), and
DIPEA (6 equiv) at RT for 2 h, then evaporation to dryness
H
DOI: 10.1021/acs.inorgchem.7b01247
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
in vacuo. A solution of TFA (97.5%) and Milli-Q water (2.5%)
was added to the crude material to deprotect the peptide,
followed by removal of the trifluoroacetic acid by sparging with
(0.1% TFA in CH CN) from 0 to 100% B over 20 min and UV
detection at λ 254 nm.
3
X-ray structure determination and refinement was obtained
1
a stream of N . The peptide was precipitated with diethyl ether,
for [ReO(HL (OMe)) ]TFA on an Oxford Diffraction Super-
2
2
isolated by centrifugation (3 min, 3600 rpm) and dissolved in
Milli-Q water (5 mL), and finally purified by semiprep RP-
HPLC (Column 1); Gradient elution of Buffer A (0.1% TFA in
H O) and Buffer B (0.1% TFA in CH CN) from 0 to 40% B to
Nova CCD diffractometer using Cu−Kα radiation, and the
temperature during data collection was maintained at 130.0(1)
using an Oxford Cryosystems cooling device. The structure was
solved by direct methods using SHELXT and refined using
2
3
−1
74,75
A, over 40 min (1.0% min ), UV detection at λ 220 nm with a
least-squares methods using SHELXL.
Thermal ellipsoid
−1
flow rate of 5 mL min .
plots were generated using ORTEP-3 integrated within the
3
76
Linear protected Tyr -octreotate peptide, dPhe-Cys(Trt)-
Tyr(tBu)-dTrp(tBoc)-Lys(tBoc)-Thr(tBu)-Cys(Trt)-Thr-
WINGX suite of programs. The trifluoroacetate counterion,
although recognizable from the difference electron density
maps, was badly disordered and could not be modeled
satisfactorily. Application of the Squeeze procedure gave a
(
tBu)−OH,was synthesized using standard automated Fmoc
SPPS procedures on a 2-chorotrityl chloride or Wang resin
unless otherwise specified.
73
3
void volume of 272 Å containing 127 electrons, consistent
77
Analytical reversed phase high performance liquid chroma-
tography (RP-HPLC) was undertaken using an Agilent 1100
with the presence of two trifluoroacetate anions per unit cell.
The charge on the complex is unambiguously (+1) given the
presence of the two pyridinium protons which are involved in
intramolecular hydrogen bonds and the ethylamino protons
which are also involved in hydrogen bonds. The crystallo-
graphic data has been deposited in the Cambridge Structural
Database (CCDC 1543360).
−1
Series HPLC system at a flow rate of 1 mL min with either
Column 1: A Zorbax Eclipse XDB-C18 column (150 mm × 4.6
mm, 5.0 μm) or Column 2: A Phenomenex Aeris Peptide XB-
C18 column (250 mm × 4.6 mm, 3.6 μm). Solvent gradients
for analytical analysis were either using System A: Gradient
1−4
elution of Buffer A (0.1% TFA in H O) and Buffer B (0.1%
Ligand Synthesis. Note: The designation of H2L refers to
the structure with a carboxylic acid-substituted, pyridylhydrazine
with the different thiocarbahydrazide functional groups (Et, tBu,
and Ph respectively). Further derivatization of the carboxylate
functional group is represented by placing the substituted group at
the carboxylic carbon in replacement of the OH group (e.g.,
2
TFA in CH CN) from 0 to 100% B over 25 min and UV
3
detection at λ 214, 254, and 280 nm, System B: Gradient
elution of Buffer A (0.1% TFA in H O) and Buffer B (0.1%
2
TFA in CH CN) from 0 to 60% B over 30 min and UV
3
detection at λ 214, 220, 254, 280, and 350 nm or System E:
1
−3
Gradient elution of Buffer A (0.1% TFA in H O) and Buffer B
H2L (OMe), denotes the substitution of a methoxy at the
2
(
0.1% TFA in CH CN) from 20 to 80% B, over 30 min and UV
carbonyl carbon to give a methyl ester.
3
detection at λ 220, 254, and 350 nm with a flow rate of 1 mL
min .
Semipreparative reversed phase high performance liquid
6-[(2-Ethylcarbamothioyl)hydrazinyl]-3-pyridinecarboxylic
−1
1
acid, H L . To a suspension of 6-hydrazino-3-pyridinecarbox-
2
2
2
ylic acid (0.45 g, 2.9 mmol) in anhydrous ethanol (5 mL) was
added ethyl isothiocyanate (0.51 mL, 5.8 mmol) under an
atmosphere of nitrogen. The suspension was heated to reflux
for 16 h then cooled to RT, and the precipitate that formed was
chromatography (semiprep RP-HPLC) was performed using an
Agilent 1200 series preparative HPLC unit with variable
wavelength detector. An automated Agilent 1200 fraction
collector collected 0.5−4 mL fractions. Peak separation was
achieved using either Column 3: Kinetex C18 100 Å, AXIA
column (150 mm × 21.2 mm, 5 μm), Column 4: Phenomenex
Synergi Hydro-RP 80 Å (50 mm × 21.2 mm, 4 μm), Column 5:
Varian Pursuit XRs C18 100 Å (150 × 21.2 mm, 5 μm) or
Column 6: SGE ProteCol C18 120 Å (250 mm × 10 mm, 5
μm). Gradient elution, flow rate, and wavelength detection are
compound specific and are detailed under the Experimental
Section of a particular compound. Each fraction collected above
collected by filtration, washed with cold ethanol (20 mL), and
1
diethyl ether (20 mL) to give H L as a colorless powder (0.67
2
1
g, 95%). H NMR [DMSO-d , 500 MHz]: δ (ppm) 9.39 (1H,
6
2
s, NH), 9.01 (1H, s, NH), 8.63 (1H, d, J = 1.7 Hz, pyH ), 8.19
(1H, t, J = 4.8 Hz, NH), 8.04 (1H, dd, J = 8.8, 2.2 Hz, pyH ),
6.53 (1H, d, J = 8.8 Hz, pyH ), 3.45 (2H, dt, J = 13.3, 6.8 Hz,
CH ), 1.03 (3H, t, J = 7.1 Hz, CH ); C{ H} NMR [DMSO-
4
5
13
1
2
3
d , 125.7 MHz]: δ (ppm) 181.5 (C, NCS), 166.5 (C, CO H),
6
2
6
2
4
162.0 (C, pyC ), 150.4 (C, pyC ), 138.8 (C, pyC ), 117.8 (C,
3
5
4
00 mAU was analyzed using ESI-MS and analytical HPLC.
Analytical HPLC traces of radiolabeled Re compounds
pyC ), 105.6 (C, pyC ), 38.4 (C, CH ), 14.6 (C, CH ); IR: ν
2 3 max
̅
1
88
−1
(cm ) 2981 (s/sh, N−H), 1605 (s/sh, CO), 1539 (s/sh),
were acquired using an Agilent 1200 LC system with in-line UV
and gamma detection (Flow-Count, LabLogic). Peak separa-
tion was achieved using an Agilent Eclipse XDB-C18 column
1319 (m/sh), 1282 (s/sh), 1245 (s/sh), 782 (s/sh); ESI-MS
+
( ): m/z calc’d for C H N O S 241.0759, found 241.0817 {[M
9
13
4
2
+
+ H] , 100%}; RP-HPLC (Column 1, System A): R (min)
T
(
4.6 × 150 mm, 5 μm), with column 1 and system F: Gradient
7.2.
elution of Buffer A (0.1% TFA in H O) and Buffer B (0.1%
TFA in CH CN) from 0 to 100% B over 20 min and UV
6-[2-(tert-Butylcarbamothioyl)hydrazinyl]-3-pyridinecar-
boxylic acid, H L . To a suspension of 6-hydrazino-3-
2
2
3
2
detection at λ 220 nm.
pyridinecarboxylic acid (0.45 g, 2.9 mmol) in anhydrous
DMA (5 mL) was added tert-butyl isothiocyanate (0.56 mL, 4.4
mmol) under an atmosphere of nitrogen. The suspension was
heated to 85 °C and after 30 min became a yellow mixture,
which was heated at 85 °C for a further 3 h. The mixture was
concentrated by evaporation under reduced pressure to a
volume of approximately 1 mL and cold diethyl ether (15 mL)
was added. The suspension was stirred vigorously overnight at
RT. The precipitate was collected via filtration and washed with
copious amounts of cold diethyl ether to afford an off-white
Analytical HPLC traces of radiolabeled 99mTc compounds
were acquired using a Shimadzu 10 AVP UV−visible
spectrophotometer (Shimadzu, Kyoto, Japan) and a sodium
iodide scintillation detector with two LC-10ATVP solvent
delivery systems for solvents A and B. Peak separation was
achieved using Column 7: Nacalai Tesque Cosomosil 5C18-AR
Waters column (4.6 × 150 mm, 5 μm) (Kyoto, Japan) at a flow
−1
rate of 1 mL min . Gradient elution followed System C:
Gradient elution of Buffer A (0.1% TFA in H O) and Buffer B
2
I
DOI: 10.1021/acs.inorgchem.7b01247
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Inorganic Chemistry
Article
1
solid (0.68 g, 86%). H NMR [MeOH-d , 500 MHz]: δ (ppm)
sat. NaCl. The organic layer was collected and dried over
4
2
8
.73 (1H, d, J = 2.2 Hz, pyH ), 8.18 (1H, dd, J = 8.7, 2.2 Hz,
pyH ), 6.76 (1H, dd, J = 8.7, 0.7 Hz, pyH ), 1.51 (9H, s);
C{ H} NMR [MeOH-d , 125.7 MHz]: δ (ppm) 181.0 (C,
NCS), 166.1 (C, CO H), 162.0 (C, pyC ), 150.4 (C, pyC ),
38.8 (C, pyC ), 117.8 (C, pyC ), 105.6 (C, pyC ), 54.7 (C,
C(CH ) ), 29.0 (3C, -C(CH ) ); IR: ν (cm ) 1604 (s/sh,
Na SO , filtered, and concentrated to afford a chalky, yellow
2
4
4
5
1
powder (0.08 g, 81%). H NMR [CHCl -d, 600 MHz]: δ
(ppm) 8.95 (1H, d, J = 2.4 Hz, pyH ), 8.21 (1H, dd, J = 8.3, 2.4
Hz, pyH ), 7.40 (1H, d, J = 8.3 Hz, pyH ), 3.92 (3H, s, OCH );
C{ H} NMR [CHCl -d, 151 MHz]: δ (ppm) 165.0 (C,
3
3
1
3
1
2
4
6
2
4
5
2
3
4
3
5
13
1
1
−1
6
2
4
-
CO Me), 155.7 (C, pyC ), 151.1 (C, pyC ), 139.7 (C, pyC ),
3
3
3 3
max
2
3
5
CO), 1533 (s/sh), 1280 (s/sh), 1252 (s/sh), 1133 (m/sh),
125.1 (C, pyC ), 124.3 (C, pyC ), 52.7 (C, OCH ); HRMS
(ESI ): m/z calc’d for C H N O 168.0773, found 168.0777
{[M + H] , 100%}; RP-HPLC (Column 2, System A): R
3
+
+
1
002 (m/sh), 779 (s/sh); HRMS (ESI ): m/z calc’d for
7
10
3
2
+
+
C H N O S 269.1072, found 269.1110 {[M + H] , 100%};
11
17
4
2
T
RP-HPLC (Column 1, System A): R (min) 10.6.
(min) 7.6.
T
6
-[2-(Phenylcarbamothioyl)hydrazinyl]-3-pyridinecarbox-
Methyl 6-[(2-Ethylcarbamothioyl)hydrazinyl]-3-pyridine-
carboxylate, H L (OMe). Crude methyl 6-hydrazinyl-3-pyridi-
3
1
ylic acid, H L . To a suspension of 6-hydrazino-3-pyridine-
2
2
carboxylic acid (0.45 g, 2.9 mmol) in anhydrous dimethylace-
tamide (DMA) (5 mL) was added phenyl isothiocyanate (0.59
mL, 4.4 mmol) under an atmosphere of nitrogen. The
suspension was heated to 65 °C and after 5 min became a
yellow solution, which was heated at 65 °C for a further 2 h.
The mixture was concentrated by evaporation under reduced
pressure to a volume of approximately 1 mL, and cold diethyl
ether (15 mL) was added A precipitate was collected by
filtration and washed with copious amounts of cold diethyl
necarboxylate, 4, (approximately 0.06 g, 0.67 mmol), was added
to anhydrous ethanol (8 mL) and heated until entirely
dissolved. Ethyl isothiocyanate (60 μL, 0.67 mmol) was
added to the solution and stirred at reflux for 3 h. The
solution was cooled to RT. A stream of N was blown over the
2
solution, and the resulting precipitate was collected by filtration
and washed with copious amounts of diethyl ether to afford a
1
colorless crystalline solid (0.05 g, 51%). H NMR [DMSO-d ,
6
400 MHz]: δ (ppm) 9.41 (1H, s, -NHNHCS), 9.10 (1H, s,
1
2
ether to afford an off-white powder (0.81 g, 97%). H NMR
-NHNHCS), 8.66 (1H, d, J = 1.8 Hz, pyH ), 8.21 (1H, br t, J =
4
[
DMSO-d , 400 MHz]: δ (ppm) 9.88 (1H, br s, NH), 9.84
6.5 Hz, -NHCH CH ), 8.06 (1H, dd, J = 8.8, 2.1 Hz, pyH ),
6
2
3
5
(
1H, br s, NH), 9.22 (1H, br s, NH), 8.67 (1H, d, J = 1.9 Hz,
6.55 (1H, d, J = 8.8 Hz, pyH ), 3.80 (3H, s, OCH ), 3.45 (2H,
3
2
4
pyH ), 8.07 (1H, d, J = 8.4 Hz, pyH ), 7.47 (2H, d, J = 7.2 Hz,
ArH), 7.30 (2H, t, J = 7.2 Hz, ArH), 7.13 (1H, t, J = 7.2 Hz,
quin., J = 6.6 Hz, CH CH ), 1.03 (3H, t, J = 7.1 Hz, CH CH );
2
3
2
3
13
1
C{ H} NMR [DMSO-d , 151 MHz]: δ (ppm) 181.3 (C,
6
5
13
1
6
2
ArH), 6.66 (1H, d, J = 8.8 Hz, pyH ); C{ H} NMR [MeOH-
NCS), 165.4 (C, CO CH ), 162.2 (C, pyC ), 150.2 (C, pyC ),
2 3
4
3
5
d , 125.7 MHz]: δ (ppm) 181.3 (C, NCS), 166.4 (C, CO H),
138.5 (C, pyC ), 116.7 (C, pyC ), 105.9 (C, pyC ), 51.9 (C,
CO CH ), 38.4 (C, CH ), 14.6 (C, CH ); IR: ν (cm ) 3143
4
2
6
2
4
−1
1
61.7 (C, pyC ), 150.3 (C, pyC ), 139.2 (C, pyC ), 138.7 (C,
2
3
2
3
max
1
3,5
4
2,6
ArC ), 127.9 (2C, ArC ), 125.6 (C, ArC ), 124.9 (2C, ArC ),
(m/br, N−H), 2977 (m/br), 1710 (s/sh, CO), 1533 (m/
3
5
−1
117.8 (C, pyC ), 106.1 (C, pyC ); IR: ν (cm ) 1607 (s/sh,
br), 1295 (s/sh), 1254 (s/sh), 1240 (m/sh), 1119 (w/sh), 781
max
+
CO), 1596 (s/sh), 1537 (s/sh), 1280 (s/sh), 1254 (s/sh),
(m/sh); HRMS (ESI ): m/z calc’d for C H N O 255.0916,
1
0
15
4
2
+
+
1
140 (m/sh), 1019 (m/sh), 782 (m/sh); HRMS (ESI ): m/z
found 255.0938 {[M + H] , 100%}; RP-HPLC (Column 2,
+
calc’d for C H N O S 289.0759, found 289.0749 {[M + H] ,
System A): R (min) 9.2.
13
13
4
2
T
1
00%}; RP-HPLC (Column 1, System A): R (min) 9.9.
Methyl 6-Chloropyridine-3-carboxylate, 3. A suspension of
Methyl 6-[(2-tert-Butyllcarbamothioyl)hydrazinyl]-3-pyri-
T
2
dinecarboxylate, H L (OMe). The compound 6-[2-(tert-
2
6
-chloronicotinic acid (5.0 g, 32 mmol) in methanol (100 mL)
butylcarbamothioyl)hydrazinyl]-3-pyridinecarboxylic acid,
2
was cooled to 0 °C, and H SO (0.5 mL) was added dropwise.
(H L ) (0.2 g, 0.74 mmol), was added to deoxygenated and
2
4
2
The mixture was heated at 60 °C for 8 h. The crude reaction
mixture was evaporated to dryness under reduced pressure. The
residue was dissolved in ethyl acetate and washed with sat.
NaHCO . The organic layer was dried over MgSO , filtered,
and evaporated to dryness in vacuo. The pale yellow solid was
recrystallized from methanol to form clear, plate-like crystals
dried CH OH (20 mL). Sulfuric acid (∼0.5 mL) was added
3
dropwise, and the reaction was heated to 50−60 °C for 3 h.
Addition of aqueous NaOH (0.1 M) caused a precipitate to
3
4
form (pH 7−8). The precipitate was collected by filtration as a
1
gray powder (0.13 g, 63%). H NMR [CHCl -d, 500 MHz]: δ
3
2
(ppm) 8.74 (1H, d, J = 1.7 Hz, pyH ), 8.16 (1H, dd, J = 8.7, 2.2
Hz, pyH ), 6.76 (1H, dd, J = 8.7, 0.3 Hz, pyH ), 3.86 (3H, s,
1
4
5
(
(
5.0 g, 91%). H NMR [CHCl -d, 400 MHz]: δ (ppm) 9.00
3
2
13
1
1H, dd, J = 2.4, 0.7 Hz, pyH ), 8.25 (1H, dd, J = 8.3, 2.4 Hz,
pyH ), 7.42 (1H, dd, J = 8.3, 0.8 Hz, pyH ), 3.96 (3H, s, CH );
C{ H} NMR [CHCl -d, 125.7 MHz]: δ (ppm) 165.0 (C,
CO CH ), 1.47 (9H, s, (CH ) ); C{ H} NMR [CH OH-d ,
2 3 3 3 3 4
4
5
3
125.7 MHz]: δ (ppm) 180.9 (C, NCS), 165.6 (C, CO CH ),
2 3
1
3
1
6
2
4
3
161.4 (C, pyC ), 150.6 (C, pyC ), 140.1 (C, pyC ), 119.8 (C,
6
2
4
3
5
CO Me), 155.8 (C, pyC ), 151.3 (C, pyC ), 139.8 (C, pyC ),
pyC ), 106.1 (C, pyC ), 53.8 (C, -OCH ), 52.1 (C, -C(CH ) ),
28.7 (3C, -C(CH ) ); IR: ν
max
2
3 3 3
3
5
−1
1
25.2 (C, pyC ), 124.4 (C, pyC ), 52.8 (C, OCH ); ESI-MS
(cm ) 1721 (s/sh, CO),
3
3 3
̅
+
(
): m/z calc’d for C H ClNO 172.0165, found 171.9965 {[M
H] , 100%}; RP-HPLC (Column 2, System A): R (min)
1523 (m/sh), 1285 (s/sh), 1250 (s/sh), 1133 (m/sh), 775 (s/
7
7
2
+
+
+
sh); ESI-MS ( ): m/z calc’d for C H N O S 283.1229, found
T
12 19
4
2
+
8.7.
283.0450 {[M + H] , 100%}; RP-HPLC (Column 2, System
Methyl 6-Hydrazinyl-3-pyridinecarboxylate, 4. Methyl 6-
A): R (min) 12.9.
T
chloropyridine-3-carboxylate, 3, (0.10 g, 0.58 mmol), and
Methyl 6-[(2-Phenylcarbamothioyl)hydrazinyl]-3-pyridine-
−
3
3
hydrazine hydrate (50−60% N H , d = 1.029 g cm , 0.10 mL,
carboxylate, H L (OMe). The same procedure and reagents
2
4
2
1
approximately 3 equiv) were added to a 5 mL microwave vial,
were used as for the synthesis of H L (OMe), except using
2
which was evacuated and purged with N . Degassed methanol
phenyl isothiocyanate (45 μL, 0.38 mmol) and methyl 6-
hydrazinyl-3-pyridinecarboxylate (63 mg, 0.38 mmol) to afford
2
(
5 mL) was added to the vial, and the suspension was subjected
1
to microwave irradiation for 15 min at 105 °C. The mixture was
filtered and concentrated in vacuo. The residue obtained was
a white, microcrystalline solid (0.08 g, 71%). H NMR
2
[CH OH-d , 400 MHz]: δ (ppm) 8.77 (1H, s, pyH ), 8.19
3
4
4
2,6
dissolved in ethyl acetate and washed with sat. NaHCO and
(1H, d, J = 8.8 Hz, pyH ), 7.51 (2H, dd, J = 8.1 Hz, ArH ),
3
J
DOI: 10.1021/acs.inorgchem.7b01247
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
.35 (2H, t, J = 7.6 Hz, ArH ), 7.21 (1H, t, ArH ), 6.85 (1H,
Article
3
,5
4
−1
7
to A, over 50 min (1.0% min ) and UV detection at λ 220 and
254 nm with a flow rate of 5 mL min . Fractions containing
5
13
1
−1
d, J = 8.8 Hz, pyH ), 3.89 (3H, s, CO CH ); C{ H} NMR
2
3
[
DMSO-d , 125.7 MHz]: δ (ppm) NCS resonance not
the desired compound were identified by HPLC-MS,
6
6
detected, 165.0 (C, CO Me), 161.6 (C, pyC ), 150.4 (C,
pyC ), 138.8 (C, ArC ), 137.7 (C, pyC ), 128.0 (2C, ArC ),
consolidated, and lyophilized to afford a colorless fluffy solid
2
2
1
4
3,5
1
(0.19 g, 27%). H NMR [MeOH-d , 600 MHz]: δ (ppm) 8.48
4
4
2,6
3
2
4
1
27.0 (C, ArC ), 124.6 (2C, ArC ), 116.6 (C, pyC ), 105.3 (C,
(1H, d, J = 2.1 Hz, pyH ), 8.29 (1H, dd, J = 9.1, 2.2 Hz, pyH ),
7.05 (1H, d, J = 9.1 Hz, pyH ), 4.38 (1H, dd, J = 8.9, 5.2 Hz,
5
−1
5
pyC ), 51.8 (C, CO CH ); IR: ν (cm ) 3262 (m/sh, N−
2
3
max
H), 1707 (s/sh, CO), 1618 (m/sh), 1537 (s/sh), 1274 (s/
αCH), 3.70 (3H, s, CO CH ), 3.38 (2H, t, J = 7.2 Hz, εCH ),
2
3
2
+
sh), 1133 (m/sh), 742 (m/sh); ESI-MS ( ): m/z calc’d for
1.97 (3H, s, COCH ), 1.87−1.84 (1H, m, βCH ), 1.75−1.69
3
2
+
C H N O S 303.0916, found 303.1274 {[M + H] , 100%};
RP-HPLC (Column 2, System A): R (min) 13.1.
(1H, m, βCH ), 1.65−1.61 (2H, m, δCH ), 1.53 (9H, s,
14
15
4
2
2
2
13 1
(CH ) ), 1.49−1.41 (2H, m, γCH ); C{ H} NMR [MeOH-
d , 150.9 MHz]: δ (ppm) 184.0 (C, NCS), 174.2 (C,
T
3 3
2
1
H L (Lys). Dichloromethane (10 mL) was added to 2-
2
4
−1
chlorotrityl resin (1.00 mmol g loading) (3.1 g, 3.1 mmol)
swelling the resin. A mixture of N -tBoc-N -Fmoc-L-Lys (1.7 g,
3
-COCH ), 173.4 (C, -CO CH ), 165.9 (C, CONH), 159.1
3
2
3
6
2
4
3
(C, pyC ), 149.4 (C, pyC ), 141.4 (C, pyC ), 123.7 (C, pyC ),
110.6 (C, pyC ), 54.9 (C, -C(CH ) ), 53.7 (C, αCH ), 52.7 (C,
α
ε
5
.6 mmol) and DIPEA (2.1 mL, 12 mmol) in dichloromethane
3
3
2
(
15 mL) was added to the resin, which was then shaken at
−CO CH ), 40.7 (C, εCH ), 32.1 (C, βCH ), 29.8 (C, δCH ),
2
3
2
2
2
ambient temperature for 2 h. The resin was filtered and the
filtrate was discarded. The resulting resin was washed with
dichloromethane (3 mL), DMF (3 mL), dichloromethane (3
mL), and finally diethyl ether (3 mL). After several hours of
suction drying the resin was weighed and loading determined
28.9 (3C, -C(CH ) ), 24.2 (C, −COCH ), 22.3 (C, γCH );
3
3
3
2
+
HRMS (ESI ): m/z calc’d for C H N O S 453.2284, found
453.2288 {[M + H] , 100%}; RP-HPLC (Column 2, System
20
33
6
4
+
A): R (min) 12.5.
T
1
H L (cRGDfK). To a solution of 6-[(2-ethylcarbamothioyl)-
2
−1
1
(
0.77 mmol g ). To the dried resin-bound tBoc-Lys(Fmoc)−
hydrazinyl]-3-pyridinecarboxylic acid, (H L ) (16 mg, 66
2
OH (0.51 g, 0.39 mmol) was added a DMF/piperidine (80:20
v/v) solution (20 mL), which was manually stirred. After 20
min, the supernatant was removed by filtration through a
sintered frit, and the remaining resin was washed with DMF (3
mL). A positive TNBSA assay (2,4,6-trinitrobenzenesulfonic
acid in methanol (5% w/v)) was used to indicate deprotection
μmol) in DMF (0.5 mL) was added HATU (25 mg, 66
μmol) and DIPEA (23 μL, 0.13 mmol). A solution of cRGDfK
(10 mg, 17 μmol) in DMF (0.3 mL) was added to the initial
mixture and then shaken for 2 h at ambient temperature.
Diethyl ether (40 mL) was added and the subsequent
suspension centrifuged (3 min, 3600 rpm). The supernatant
was discarded and the process repeated. The remaining solid
was dissolved in a H O/CH CN (90:10 v/v) solution, filtered
of the Fmoc protected epsilon (ε) amine. A mixture of HATU
1
(
285 mg, 0.75 mmol), H L (176 mg, 0.75 mmol), and DIPEA
2
2
3
(
261 μL, 1.5 mmol) in DMF (4 mL) was added to the resin
(Millipore 0.45 μm porosity syringe filter), and then purified by
and reacted for 4 h at ambient temperature. The liquid was
drained and the resin was washed successively with dichloro-
methane (3 mL), then DMF (3 mL), and again with
semipreparative RP-HPLC (Column 4). The HPLC system
involved a gradient elution of Buffer A (0.1% TFA in H O) and
2
Buffer B (0.1% TFA in CH CN) from 0 to 50% B to A, over 65
3
−1
dichloromethane (3 mL). A TFA/H O/dichloromethane
min (1.3% min ) and UV detection at λ 220, 254, 275, and
2
−1
(
(
18:2:80 v/v) solution was added to half the resin
approximately 0.19 mmol), and the mixture shaken for 1 h
350 nm with a flow rate of 8 mL min . Fractions containing
the desired compound were identified by HPLC-MS,
consolidated and lyophilized to afford a colorless solid (10
mg, 80%). ESI-MS ( ) m/z calc’d for [C H N O S]
826.378, found 826.376, calc’d for [C H N O S]
and then filtered. The filtrate was concentrated in vacuo and
diethyl ether (40 mL) was added to form a suspension, which
was centrifuged (3 min, 3600 rpm) and the supernatant was
discarded. The crude material was dissolved in H O/CH CN
+
+
+
3
6
52 13
8
2
3
6
53 13
8
413.693, found 413.692; RP-HPLC (Column 2, System A):
2
3
(
90:10 v/v), filtered (Millipore 0.45 μm porosity syringe filter),
R (min) 11.3.
T
2
and then purified by semipreparative RP-HPLC (Column 5).
The HPLC system involved a gradient elution of Buffer A
H L (cRGDfK). The same procedure was used as for
2
1
1
H L (cRGDfK), except H L was replaced with 6-[(2-tert-
2
2
(
2
0.1% TFA in H O) and Buffer B (0.1% TFA in CH CN) from
butylcarbamothioyl)hydrazinyl]-3-pyridinecarboxylic acid,
2
3
−1
2
0 to 40% B to A, over 20 min (1.0% min ) then 40−60%
(H L ) (5.3 mg, 19 μmol), and other reagent quantities were
2
−1
over 40 min (0.5% min ) and UV detection at 220 nm with a
flow rate of 5 mL min . Fractions containing the desired
compound were identified by HPLC-MS, consolidated and
adapted accordingly; HATU (6.5 mg, 17 μmol), DIPEA (45
μL, 0.13 mmol), and cRGDfK (10 mg, 17 μmol). Semi-
preparative RP-HPLC purification (Column 4) involved a
−1
lyophilized to afford an off-white, fluffy compound (10 mg,
gradient elution of Buffer A (0.1% TFA in H O) and Buffer B
2
+
1
4%). ESI-MS ( ): m/z calc’d for C H N O S 369.1709,
(0.1% TFA in CH CN) from 0 to 40% B to A, over 80 min
1
5
25
6
3
3
+
−1
found 369.1798 {[M + H] , 100%}; RP-HPLC (Column 2,
(0.5% min ) and UV detection at λ 220, 254, 275, and 350 nm
−
1
System A): R (min) 6.00, (Column 2, System B) 24.0.
with a flow rate of 8 mL min . Fractions containing the desired
compound were identified by HPLC-MS, consolidated and
lyophilized to afford a light yellow solid (3.4 mg, 24%). ESI-MS
( ) m/z calc’d for [C H N O S] 854.4096, found 854.4097,
calc’d for [C H N O S] 427.7087, found 427.7083; RP-
38 57 13 8
T
2
H L (N OAc-Lys-OMe). A solution of HATU (0.23 g, 0.56
2
α
mmol) and DIPEA (0.29 mL, 1.1 mmol) in DMSO (1 mL) was
2
+
+
added to H L (0.15 g, 0.56 mmol) and shaken for 2 min. N -
2
α
38 56 13 8
2+
Acetyl-L-lysine methyl ester hydrochloride (0.10 g, 0.42 mmol)
in DMSO (1 mL) was added and shaken for 2 h. Diethyl ether
HPLC (Column 2, System A): R (min) 12.9.
T
3
(
40 mL) was added and the mixture was centrifuged. The
H L (cRGDfK). The same procedure was used as for
H L (cRGDfK), except H L was replaced with 6-[(2-
2
1
1
supernatant was discarded and the remaining yellow oil was
purified by semipreparative RP-HPLC (Column 5). The HPLC
system involved a gradient elution of Buffer A (0.1% TFA in
H O) and Buffer B (0.1% TFA in CH CN) from 10 to 60% B
2
2
phenylcarbamothioyl)hydrazinyl]-3-pyridinecarboxylic acid,
3
(H L ) (5.2 mg, 19 μmol) and other reagent quantities were
2
adapted accordingly; HATU (6.8 mg, 18 μmol), DIPEA (45
2
3
K
DOI: 10.1021/acs.inorgchem.7b01247
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
μL, 0.13 mmol), and cRGDfK (10 mg, 17 μmol). Semi-
preparative RP-HPLC purification (Column 4) involved a
containing the desired compound were identified by HPLC-
MS, consolidated and lyophilized to afford a pale yellow solid
+
+
+
gradient elution of Buffer A (0.1% TFA in H O) and Buffer B
(56 mg, 18%). HRMS ( ) m/z calc’d for [C H N O S ]
2
58 75 14 13 3
2
(
0.1% TFA in CH CN) from 0 to 40% B to A, over 80 min
1271.4800, found 1271.4760, calc’d for [C H N O S ]
58 75 14 13 3
3
−1
(
0.5% min ) and UV detection at λ 214, 220, and 254 nm with
636.2439, found 636.2416; RP-HPLC (Column 2, System A):
−1
a flow rate of 8 mL min . Fractions containing the desired
compound were identified by HPLC-MS, consolidated, and
lyophilized to afford a colorless solid (8.6 mg, 55%). ESI-MS
R (min) 13.0.
T
2
3
H L (Tyr -Oct). The same procedure was used as for
2
1
3
1
2
H L (Tyr -Oct), except H L was replaced with H L (0.20
2
2
2
+
+
(
) m/z calc’d for [C H N O S] 874.3783, found 874.3380,
g, 0.75 mmol, 3 equiv), and all other reagents were used
according to their reported equivalencies. The precipitate after
lyophilization was dissolved in CH CN/H O (8 mL, 50:50 v/v)
4
0
52 13
8
2
+
calc’d for [C H N O S] 437.6930, found 437.6992; RP-
40
53 13
8
HPLC (Column 2, System A): R (min) 14.2.
T
3
2
1
3
3
H L (Tyr -Oct). Linear protected Tyr -octreotate peptide
and filtered (Millipore 0.45 μm porosity syringe filter). The
compound was purified by semipreparative RP-HPLC (Col-
umn 5) with an isocratic step gradient system of Buffer A (0.1%
TFA in H O) and Buffer B (0.1% TFA in CH CN). The
2
(
dPhe-Cys(Acm)-Tyr(tBu)-dTrp(tBoc)-Lys(tBoc)-Thr(tBu)-
Cys(Acm)-Thr(tBu)-OH) was prepared manually by standard
Fmoc SPPS on Wang resin preloaded with H-Thr(tBu)−OH
2
3
−1
(
100 mg, 0.5 mmol g ). Amino acid coupling: Coupling of
elution method involved 28% B for 10 min then 32% B for 40
each amino acid was achieved by addition to the resin of a
premade solution of Fmoc-amino acid (2 mmol, 4 equiv),
HATU (1.9 mmol, 3.8 equiv), and DIPEA (4 mmol, 8 equiv) in
DMF (5 mL). The resin mixture was shaken in a reaction vessel
equipped with a sintered bottom for 1 h then filtered and
washed with DMF (3 mL), dichloromethane (3 mL), and again
with DMF (3 mL). Successful coupling was confirmed by a
negative TNBSA test. Fmoc deprotection: Cleavage of the
Fmoc groups was achieved after each successive coupling
reaction. A DMF/piperidine (80:20 v/v) solution was added to
the protected resin, which was contained in a reaction vessel
with a sintered frit. The mixture was stirred for 5 min, and then
the resin was filtered and washed with cleavage solution, then
DMF (2 mL), dichloromethane (2 mL) and again DMF (3
mL). Complete deprotection was confirmed with a positive
TNBSA test. Acm-group deprotection and on-resin cyclization:
After the final Fmoc deprotection of resin-bound Fmoc-dPhe-
OH, thallium(III) trifluoroacetate (Tl(CF CO ) ) (0.54 g, 1.0
min (desired peak at 34 min) and UV detection at λ 214, 220,
−1
254, 280, and 350 nm with a flow rate of 8 mL min . Fractions
containing the desired compound were identified by HPLC-
MS, consolidated, and lyophilized to afford a fluffy, colorless
+
compound (18 mg, 5.5%). HRMS ( ) m/z calc’d for
+
[C H N O S ] 1299.5113, found 1299.5110, calc’d for
60
79 14 13 3
2
+
[C H N O S ] 650.2596, found 650.2599; RP-HPLC
60
80 14 13 3
(Column 2, System A): R (min) 14.4.
T
3
3
H L (Tyr -Oct). The same procedure was used as for
2
1
3
1
3
H L (Tyr -Oct), except H L was replaced with H L (0.22
2
2
2
g, 0.75 mmol, 3 equiv). The precipitate after lyophilization was
dissolved in CH CN/H O (10 mL, 50:50 v/v) and filtered
3
2
(Millipore 0.45 μm porosity syringe filter). The compound was
purified by semipreparative RP-HPLC (Column 5) with a
linear gradient system of Buffer A (0.1% TFA in H O) and
2
Buffer B (0.1% TFA in CH CN) from 20% to 50% B to A over
3
−
1
60 min (0.5% min ) (or isocratic elution at 29% Buffer B) and
−1
UV detection at λ 254 nm with a flow rate of 7 mL min .
Fractions containing the desired compound were identified by
HPLC-MS, consolidated and lyophilized to afford a fluffy white
solid (14 mg, 4.2%). HRMS ( ) m/z calc’d for
[C H N O S ] 1319.4800, found 1319.4849, calc’d for
3
2 3
mmol, 2 equiv) in DMF (4 mL) was added to the resin, and the
mixture was stirred for 2 h. The liquid was decanted from the
resin and the resin was washed with DMF (15 mL),
dichloromethane (3 mL), and diethyl ether (2 mL). SHYNIC
+
+
6
2
75 14 13 3
2+
coupling: Half the resin-bound peptide (approximately 0.25
[C H N O S ] 650.2596, found 636.2425; RP-HPLC
62 75 14 13 3
1
mmol) was used for the following reaction. H L (0.18 g, 0.75
(Column 2, System A): R (min) 14.1.
2
T
mmol, 3 equiv) was dissolved in DMF (3 mL) and HATU
Synthesis of Rhenium Complexes. Note: Unless specified
(
0.29 g, 0.75 mmol, 3 equiv) added to preactivate the ligand.
Re represents ‘rhenium with natural isotope abundance’.
1
1
DIPEA (0.26 mL, 1.5 mmol, 6 equiv) was added and the
mixture shaken for 2 min until full dissolution had occurred.
The yellow solution was added to the dry resin in a reaction
vessel equipped with a sintered frit and shaken for 2 h. The
reaction solution was drained and the resin was washed with
DMF (15 mL), dichloromethane (3 mL) and again DMF (3
mL). Resin and acid-sensitive protecting group cleavage: The
[ReO(HL (OMe)) ]Cl. To H L (OMe) (90 mg, 0.35 mmol)
2 2
and (tBu N)[ReOCl ] (103 mg, 0.18 mmol) was added
4
4
anhydrous MeOH (13 mL). The mixture immediately turned
deep red and was stirred at room temperature for 4 h. The
reaction mixture was filtered, diethyl ether (ca. 15 mL) was
added, and the resulting precipitate was collected by filtration
1
to give [ReO(HL (OMe)) ]Cl as a dark-red, microcrystalline
2
1
petide-loaded resin was treated with a TFA/TIPS/H O
solid (0.11 g, 84%). H NMR [DMSO-d , 400 MHz]: δ (ppm)
8.63 (2H, s, pyH ), 8.26 (2H, dd, J = 9.3, 1.8 Hz, pyH ), 7.87
(2H, d, J = 9.4 Hz, pyH ), 7.43 (2H, br m, NH), 3.89 (6H, s,
2
6
2
5
(
96:1.5:2.5 v/v/v) solution (20 mL) and shaken for 2 h. The
4
mixture was filtered and sparged with a steady stream of N to
2
reduce the volume by 90%. Cold diethyl ether (40 mL) was
added to precipitate the peptide that was isolated by
centrifugation (3 min, 3600 rpm). The crude material was
dissolved in CH CN/H O (50:50 v/v) and lyophilized. The
OCH ), 3.47 (4H, qd, J = 13.6, 7.0 Hz, CH CH ), 1.16 (6H, t, J
3
2
3
13
1
= 7.5 Hz, CH CH ); C{ H} NMR [CH CN-d , 150.9 MHz]:
2
3
3
3
δ (ppm) 173.7 (2C, CO CH ), 165.1 (2C, NCS), 162.5 (2C,
2
3
6
5
2
4
pyC ), 140.5 (2C, pyC ), 139.9 (2C, pyC ), 123.9 (2C, pyC ),
122.0 (2C, pyC ), 52.3 (2C, CO CH ), 42.6 (2C, CH CH ),
3
2
3
solid was dissolved in CH CN/H O (40:60 v/v) and filtered
3
2
2
3
2
3
−
1
(
Millipore 0.45 μm porosity syringe filter). The compound was
14.5 (2C, CH CH ); IR: ν (cm ) 1553 (s/sh, CO), 1287
2
3
max
+
purified by semipreparative RP-HPLC (Column 5) with a
(s/sh), 963 (m/sh, ReO), 764 (m/sh); HRMS (ESI ): m/z
+
linear gradient elution of Buffer A (0.1% TFA in H O) and
calc’d for ReC H N O S 709.0916, found 709.0898 {[M] ,
2
20 26
8
5 2
Buffer B (0.1% TFA in CH CN) from 20 to 80% B to A, over
100%}; RP-HPLC (Column 2, System A): R (min) 14.8.
Histidine and Cysteine Challenge Experiments. A 50-fold
excess of histidine or cysteine was added to a solution of
3
T
−1
1
2
20 min (0.5% min ) and UV detection at λ 214, 220, 254,
80, and 350 nm with a flow rate of 8 mL min . Fractions
−1
L
DOI: 10.1021/acs.inorgchem.7b01247
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
1
+
[
ReO(HL (OMe)) ] (1 mg/L) in PBS Buffer (10 mM, pH
CO CH ), 3.43 (4H, q, J = 6.6 Hz, εCH ), 1.99 (6H, s,
2
2
3
2
7
.4, 4 mL) and the mixture was heated to 37 °C. At 2, 4, and 24
COCH ), 1.92−1.86 (4H, m, βCH ), 1.78−1.72 (4H, m,
3
2
h after initiation of the experiment, 10 μL aliquots of the
reaction mixture were diluted with 90 μL of Milli-Q water and
analyzed using analytical HPLC methods. The HPLC traces
showed little or no decomposition (<5%) of the rhenium
δCH ), 1.71−1.66 (4H, m, γCH ), 1.50 (18H, s, (CH ) );
2
2
3 3
13 1
C{ H} NMR [MeOH-d , 150.9 MHz]: δ (ppm) 174.2 (2C,
4
COCH ), 173.4 (2C, CO CH ), 167.2 (2C, N = CS), 165.1
3
2
3
6
5
(2C, CONH), 163.3 (2C, pyC ), 140.7 (2C, d, pyC ), 139.1
2
4
3
complex at all the time-points.
(2C, d, pyC ), 124.5 (2C, pyC ), 122.6 (2C, pyC ), 55.3 (2C,
-C(CH ) ), 53.7 (C, αCH ), 52.7 (C, −CO CH ), 40.8 (2C,
2
[
ReO(HL (OMe)) ]BPh . To a stirred suspension of trans-
2
4
3 3
2
2
3
[
ReOCl (PPh ) ] (0.15 g, 0.17 mmol) in MeOH (10 mL) was
εCH ), 32.1 (2C, βCH ), 29.9 (2C, δCH ), 28.9 (6C,
3
3 2
2 2 2
2
added H L (OMe) (0.10 g, 0.35 mmol) and 2 drops of Et N.
-C(CH ) ), 24.3 (2C, COCH ), 22.3 (2C, γCH ); HRMS
3 3 3 2
2
3
+
+
The mixture was heated at reflux for 16 h, then filtered, and
diethyl ether was added to the filtrate. The resulting precipitate
was collected by filtration, washed with cold diethyl ether, and
then dissolved in MeOH. Addition of excess NaBPh resulted
in the precipitation of a red-brown precipitate that was
(ESI ) m/z calc’d for [ReC H N O S ] 1105.3762, found
40 62 12 9 2
2+
1105.4079, calc’d for [ReC H N O S] 553.1920, found
40
62 12
9
553.2094; RP-HPLC (Column 2, System A): R (min) 16.9.
T
1
+
1
4
[ReO(HL (Lys)) ] . To resin-bound H L (Lys) (approxi-
2
2
mately 0.19 mmol) was added trans-[ReOCl (PPh ) ] (78
3 3 2
collected, and washed with hot hexane (10 mL) to afford
mg, 0.09 mmol) suspended in DMF (5 mL). The slurry was
stirred for 2 h. The green suspension turned colorless and the
resin turned dark red. The red resin was isolated by filtration
and washed with DMF (3 mL), dichloromethane (3 mL), and
again with DMF (3 mL). The rhenium complex was cleaved
from the resin by treatment with TFA/dichloromethane (50:50
v/v) solution (30 mL) for 3 h. The mixture was sparged with
2
1
[
ReO(HL (OMe)) ]BPh (72 mg, 39%). H NMR [CHCl -d,
2
4
3
2
600 MHz]: δ (ppm) 8.10 (2H, d, J = 9.5 Hz, pyH ), 7.75 (2H,
5
4
d, J = 9.4 Hz, pyH ), 7.62 (8H, m, BArH), 7.33 (2H, m, pyH ),
6
3
.93 (8H, t, J = 7.1 Hz, BArH), 6.72 (4H, t, J = 7.1 Hz, BArH),
.86 (6H, s, CO CH ), 1.39 (18H, s, (CH ) ); C{ H} NMR
1
3
1
2
3
3 3
[
(
1
CHCl -d, 150.9 MHz]: δ (ppm) 164.5, 142.5, 135.8−135.8
3
8C, BArC), 133.9−133.8, 128.9, 128.6, 128.5, 127.5, 126.2−
26.1 (8C, BArC), 122.0 (4C, BArC), 54.9 (2C, -OCH ), 52.6
N to reduce the volume to 10% of the original volume. Cold
2
3
diethyl ether (40 mL) was added, the mixture was centrifuged
(3 min, 3600 rpm), and the supernatant was discarded. This
process was repeated twice. The precipitate was dissolved in
−
1
(
(
2C, -C(CH ) ), 29.0 (6C, -C(CH ) ); IR: ν (cm ) 1555
s/sh, CO), 1284 (s/sh), 960 (m/sh, ReO), 756 (m/sh);
3 3 3 3 max
̅
+
HRMS (ESI ): m/z calc’d for ReC H N O S 765.1651,
CH CN (1.0 mL) and then Milli-Q water (2.0 mL) was added.
2
4
34
8
5
2
3
+
found 765.1559 {[M] , 100%}; RP-HPLC (Column 2, System
A): R (min) 15.0.
The complex was purified by semipreparative RP-HPLC
purification (Column 5) involved a gradient elution of Buffer
A (0.1% TFA in H O) and Buffer B (0.1% TFA in CH CN)
T
3
[
ReO(HL (OMe)) ]Cl. The same procedure was used as for
2
2
3
1
+
1
−1
[
ReO(HL (OMe)) ] , except H L (OMe) was replaced with
from 10 to 70% B to A, over 120 min (0.5% min ) and UV
2
2
3
H L (OMe) (0.10 g, 0.33 mmol). All other reagents quantities
were used accordingly; (tBu N)[ReOCl ] (95 mg, 0.16 mmol)
detection at λ 214, 220, and 254 nm, with a flow rate of 8 mL
2
−
1
4
4
min . Fractions containing the desired compound were
and MeOH (20 mL). The reaction afforded a red microcrystal-
identified by HPLC-MS, consolidated, and lyophilized to afford
1
1
line solid (69 mg, 51%). H NMR [DMSO-d , 400 MHz]: δ
a red, fluffy solid (22 mg, 23%, assuming [ReO(HL (Lys)) ]-
6
2
2
1
(
9
ppm) 9.84 (2H, s, NH), 8.79 (2H, s, pyH ), 8.42 (2H, dd, J =
CF CO ). H NMR [MeOH-d , 600 MHz]: δ (ppm) 8.58 (2H,
s, pyH ), 8.34 (2H, dd, J = 9.5, 1.8 Hz, pyH ), 7.97 (2H, d, J =
9.5 Hz, pyH ), 3.80 (2H, t, J = 6.1 Hz, αCH), 3.60 (4H, dtd, J =
3
2
4
5
4
2
5
.1, 1.7 Hz, pyH ), 8.03 (2H, d, J = 9.2 Hz, pyH ), 7.73 (4H, d,
2,6
3,5
4
J = 7.8 Hz, ArH ), 7.32 (4H, t, J = 7.9 Hz, ArH ), 6.95 (2H, t,
J = 7.3 Hz, ArH ), 3.92 (6H, s, CO CH ); C{ H} NMR
4
13
1
20.2, 13.2, 7.0 Hz, −CH CH ), 3.46 (4H, dd, J = 8.9, 5.1 Hz,
2
3
2
3
[
1
1
DMSO-d , 150.9 MHz]: δ (ppm) CO Me signal not detected,
εCH ), 1.99−1.92 (4H, m, βCH ), 1.72 (4H, dt, J = 14.5, 7.3
6
2
2
2
5
2
64.2 (2C), 145.5 (2C), 142.2 (2C, pyC ), 139.5 (2C, pyC ),
Hz, δCH ), 1.60−1.50 (4H, m, γCH ), 1.27 (6H, t,
2
2
3
,5
4
1
13 1
30.0 (2C, ArC ), 129.3 (4C, ArC ), 128.9 (2C, ArC ), 122.3
−CH CH ); C{ H} NMR [MeOH-d , 150.9 MHz]: δ
2
3
4
4
3
2,6
(
2C, pyC ), 121.4 (2C, pyC ), 117.6 (4C, ArC ), 52.6 (2C,
(ppm) 172.8 (2C, CO H), 169.0 (2C, NCS), 165.4 (2C,
2
−1
6
5
CO CH ) ); IR: ν (cm ) 1557 (s/sh, CO), 1282 (s/sh),
CONH), 163.6 (2C, pyC ), 140.5 (2C, pyC ), 138.9 (2C,
2
3 3
̅
max
+
2
4
3
9
60 (m/sh, ReO), 752 (m/sh); HRMS (ESI ): m/z calc’d
pyC ), 123.9 (2C, pyC ), 122.5 (2C, pyC ), 54.9 (2C, αCH),
42.5 (2C, CH CH ), 40.6 (2C, εCH ), 31.5 (2C, βCH ), 29.9
+
for ReC H N O S , 805.1014, found 805.1004 {[M] , 100%};
28
26
8
5
2
2
3
2
2
RP-HPLC (Column 2, System A): R (min) 15.4.
(2C, δCH ), 23.4 (2C, γCH ), 14.9 (2C, CH CH ); ESI-MS
T
2 2 2 3
2
+
2
+
+
[
ReO(HL (N -Ac-Lys-OMe)) ] . To H L (N -Ac-Lys-OMe)
( ) m/z calc’d for [ReC H N O S ] 937.2611, found
30 46 12 7 2
α
2
2
α
2
+
(
15 mg, 33 μmol) in MeOH (1 mL) was added a solution of
tBu N)[ReOCl ] (0.9 mg, 15 μmol) in MeOH (0.3 mL). The
937.2574, calc’d for [ReC H N O S ] 469.1345, found
30 47 12 7 2
3+
(
469.1355, calc’d for [ReC H N O S ] 313.0256, found
4
4
30 48 12
7 2
mixture was stirred at ambient temperature for 2 h, then diluted
with Milli-Q water and purified by semipreparative RP-HPLC
313.0939; RP-HPLC (Column 2, System A): R (min) 10.1.
T
1
+
1
[ReO(HL (cRGDfK)) ] . A solution of H L (cRGDfK) (5 mg,
2 2
(
Column 4). The HPLC system involved a gradient elution of
6.1 μmol) in MeOH (250 μL) was added to a mixture of
[tBu N][ReOCl ] (1.8 mg, 3.0 μmol) in MeOH (250 μL). The
Buffer A (0.1% TFA in H O) and Buffer B (0.1% TFA in
CH CN) from 10 to 20% B to A, over 10 min (1.0% min ),
then 20 to 40% B to A over 40 min (0.5% min ) and UV
detection at λ 214, 220, 254, and 280 nm with a flow rate of 8
mL min . Fractions containing the desired compound were
identified by HPLC-MS, consolidated and lyophilized to afford
a red solid (5 mg, 27%, assuming [ReO(HL (N -Ac-Lys-
OMe)) ]CF CO ). H NMR [MeOH-d , 600 MHz]: δ (ppm)
2
4
4
−1
mixture was shaken for 2.5 h, then diluted with Milli-Q water (5
mL), filtered (Millipore 0.45 μm porosity syringe filter), and
purified by semiprep RP-HPLC (Column 6). The HPLC
system involved isocratic elution of 77% Buffer A (0.1% TFA in
3
−
1
−1
−1
H O) and 23% Buffer B (0.1% TFA in CH CN) (0.5% min )
2
3
2
and UV detection at λ 214, 220, 254, 280, and 350 nm with a
α
1
−1
flow rate of 5 mL min . Fractions containing the desired
2
3
2
4
2
5
8
.68 (2H, s, pyH ), 8.36 (2H, d, J = 9.6 Hz, pyH ), 8.03 (2H, d,
compound were identified by HPLC-MS, consolidated, and
lyophilized to afford a white solid (3.1 mg, 53%, assuming
4
J = 9.5 Hz, pyH ), 4.43−4.40 (2H, m, αCH), 3.72 (6H, s,
M
DOI: 10.1021/acs.inorgchem.7b01247
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
1
+
[
ReO(HL (cRGDfK)) ]CF CO ). HRMS (ESI ) m/z calc’d
TFA in H O) and Buffer B (0.1% TFA in CH CN) at 38%
2
3
2
2
3
2
+
−1
for [ReC H N O S ] 926.3413, 926.3433, calc’d for
Buffer B, over 60 min (1.0% min ) and UV detection at λ 214,
72
101 26 17 2
3
+
−1
[
ReC H N O S ] 617.8968, found 617.9939; RP-HPLC
220, and 254 nm, with a flow rate of 4 mL min . Fractions
72
102 26 17 2
(
Column 2, System A): R (min) 11.0.
containing the desired compound were consolidated and
T
2
+
[
ReO(HL (cRGDfK)) ] . The same procedure was used as for
lyophilized to afford a red, fluffy material (5.5 mg, ∼61%,
2
1
+
1
2
3
+
[
ReO(HL (cRGDfK)) ] , except H L (cRGDfK) was replaced
assuming [ReO(HL (Tyr -Oct)) ]CF CO ). ESI-MS ( ) m/z
2
2
2 3 2
2
2+
with H L (cRGDfK) (8.0 mg, 9.4 μmol) in MeOH (250 μL);
calc’d for [ReC120H154N O S ]
1399.4720, calc’d for [ReC120H156N O S ] 933.6548,
1399.9784, found
2
28 27 6
3
+
(tBu N)[ReOCl ] (2.7 mg, 4.7 μmol) in MeOH (500 μL). The
4
4
28 27 6
compound was purified by semipreparative RP-HPLC
purification (Column 4) involved gradient elution of Buffer A
found 933.6460; RP-HPLC (Column 2, System A): R (min)
T
16.2.
3
3
+
(
0
4
0.1% TFA in H O) and Buffer B (0.1% TFA in CH CN) from
[ReO(HL (Tyr -Oct)) ] . Preloaded, resin-bound (Wang)
ligand, H L (Tyr -Oct) (approximately 6.0 μmol), was swollen
2
3
2
−1
3
3
to 20% over 20 min (1% min ), then 20 to 60% B to A over
2
−1
0 min (0.5% min ) and UV detection at 214, 220, 254, 280,
in CH Cl and drained twice. Anhydrous DMF (2 × 10 mL)
2
2
−1
and 350 nm with a flow rate of 8 mL min . Fractions
containing the desired compound were identified by HPLC-
MS, consolidated and lyophilized to afford a white solid (5.1
mg, ∼54%, assuming [ReO(HL (cRGDfK)) ]CF CO ).
HRMS (ESI ) m/z calc’d for [ReC H N O S ]
was added to the resin the mixture was stirred and drained.
DMF (1 mL) was added to the washed resin and (tBu N)-
4
[ReOCl ] (2.1 mg, 2.9 μmol) in DMF (50 μL) was added to
4
2
the resin. The resin mixture was reacted for 2 h at RT. The
solution was drained and the resin was washed with copious
amounts of DMF (5 × 10 mL), CH Cl (3 × 10 mL), and
2
3
2
+
2+
7
6
109 26 17 2
3+
9
6
54.3732, found 954.3715, calc’d for [ReC H N O S ]
76 110 26 17 2
2
2
36.5847, found 636.5837; RP-HPLC (Column 2, System A):
diethyl ether (10 mL). The crude material was cleaved off the
resin by addition of TFA/CH Cl /TIPS/H O (50:46:1:3 v/v)
R (min) 12.5.
T
2
2
2
3
+
[
ReO(HL (cRGDfK)) ] . The same procedure was used as for
(20 mL). The mixture was shaken for 1.5 h and filtered and the
2
1
+
1
[
ReO(HL (cRGDfK)) ] , except H L (cRGDfK) was replaced
filtrate sparged with a stream of N until the red mixture had
2
2
2
3
with H L (cRGDfK) (4.0 mg, 4.6 μmol) in MeOH (200 μL),
reduced to approximately 0.5 mL. Diethyl ether (2 × 10 mL)
was added to the crude material, and the resulting precipitate
was collected via centrifugation (3 min, 3600 rpm). The
compound was purified by semipreparative RP-HPLC
purification (Column 6) by gradient elution of Buffer A
(0.1% TFA in H O) and Buffer B (0.1% TFA in CH CN) from
2
and other reagent quantities were adapted accordingly,
[
tBu N][ReOCl ] (1.3 mg, 2.3 μmol) in MeOH (200 μL).
4 4
The HPLC system (Column 6) involved a linear gradient
elution of Buffer A (0.1% TFA in H O) and Buffer B (0.1%
2
−1
TFA in CH CN) from 0 to 20% over 20 min (1% min ), then
0 to 60% B to A over 40 min (0.5% min ) and UV detection
3
2
3
−1
−1
2
10 to 80% B to A, over 70 min (1.0% min ) and UV detection
−
1
at 214, 220, 254, 280, and 350 nm with a flow rate of 8 mL
at λ 214, 220, and 254 nm, with a flow rate of 4 mL min .
Fractions containing the desired compound were consolidated
and lyophilized to afford a red, fluffy material (5.0 mg, ∼58%,
assuming [ReO(HL (Tyr -Oct))
calc’d for [ReC124
1419.9281, calc’d for [ReC124
−
1
min . Fractions containing the desired compound were
identified by HPLC-MS, consolidated, and lyophilized to afford
a white solid (2.0 mg, ∼42%, assuming [ReO-
3
3
+
]CF CO
). ESI-MS ( ) m/z
1419.9470, found
2
3 2
3
+
2+
(
HL (cRGDfK)) ]CF CO ). HRMS (ESI ) m/z calc’d for
H N O S ]
148 28 27 6
2
3
2
2
+
3+
[
[
(
ReC H N O S ] 974.3419, found 974.3711, calc’d for
ReC H N O S ] 649.8972, found 650.0023; RP-HPLC
Column 2, System A): R (min) 12.9.
ReO(HL (Tyr -Oct)) ] . A solution of H L (Tyr -Oct) (9.0
H
149
N
28
O
27
S
6
]
946.9673,
(min)
8
0
101 26 17 2
3
+
found 946.1960; RP-HPLC (Column 2, System A): R
80
102 26 17 2
T
16.2.
T
1
3
+
1
3
188
1
3
]+. Rhenium-188 was
[
Synthesis of [ ReO(HL (Tyr -Oct))
2
2
2
produced from an ITG 1 W/ Re generator (ITG Isotope
88
188
mg, 7.1 μmol) in MeOH (50 μL) was added to a mixture of
tBu N)[ReOCl ] (2.1 mg, 3.5 μmol) and MeOH (100 μL).
(
Technologies, Garching, Germany). Generator-produced
4
4
188
−
The reaction mixture was shaken vigorously for 2 h. Milli-Q
ReO4 (86 MBq) in aqueous sodium chloride solution
water was added (4 mL) and the reaction was filtered
(200 μL, 0.9% w/v) was added to a sealed, N -purged vial
2
(
Millipore 0.45 μm porosity syringe filter). The compound
was purified by semipreparative RP-HPLC purification
Column 6) with a gradient elution of Buffer A (0.1% TFA
in H O) and Buffer B (0.1% TFA in CH CN) from 10 to 80%
containing sodium tartrate (0.2 mg) and stannous chloride (0.1
mg) in water (400 μL), followed by heating at 80 °C for 30
min. An aliquot of this solution (50 μL) was added to a
(
1
3
separate sealed, N -purged vial containing H L (Tyr -Oct)
2
3
2
2
−1
B to A, over 70 min (1.0% min ) and UV detection at λ 214,
2
containing the desired compound were identified by HPLC-
MS, consolidated, and lyophilized to afford a red, fluffy material
(100 μg) dissolved in water (100 μL). The reaction was left for
30 min at ambient temperature, after which an aliquot was
removed for HPLC analysis, revealing that there was no
reaction between 1 Re and peptide. The reaction vial was then
heated at 100 °C for 1 h, after which an aliquot was analyzed by
HPLC (column 1, system F). Radiochemical yield: 67%,
−1
20, and 254 nm, with a flow rate of 5 mL min . Fractions
88
1
3
(
6.8 mg, ∼ 68%, assuming [ReO(HL (Tyr -Oct)) ]CF CO ).
2
3
2
+
2
+
ESI-MS ( ) m/z calc’d for [ReC
H N O S ] 1371.9470,
1
16 148 28 27 6
3
+
188
1
3
+
found 1372.0024, calc’d for [ReC116H149N O S ] 914.9673,
found 915.0145; RP-HPLC (Column 2, System A): R (min)
1
retention time of [ ReO(HL (Tyr -Oct)) ] = 11.6 min,
28
27
6
2
nat
1
3
+
compared to retention time of [ ReO(HL (Tyr -Oct)) ] =
2
11.3 min and H L (Tyr -Oct) = 9.7 min.
T
1
3
5.4.
2
2
3
+
[
ReO(HL (Tyr -Oct)) ] . The same procedure was used as for
Synthesis of Technetium-99m Complexes. Sodium
2
1
3
+
1
3
99m
[
ReO(HL (Tyr -Oct)) ] , except H L (Tyr -Oct) was re-
pertechnetate, Na[ TcO ] (1000 MBq), was eluted from a
2
2
4
2
3
99
99m
placed with H L (Tyr -Oct) (8.0 mg, 6.2 μmol) in MeOH
(
Gentech Mo/ Tc sterile generator (Austin Health: Nuclear
Medicine and Centre for PET, Australia, via ANSTO Health)
2
200 μL) and other reagent quantities were adapted
accordingly: (tBu N)[ReOCl ] (1.8 mg, 3.1 μmol) in MeOH
as a 1.0 mL saline solution (0.9% v/v). A solution of SnCl in
0.1 M HCl (0.5 mg mL ) was prepared and purged with
nitrogen. Disodium tartrate dihydrate was dissolved in water (1
4
4
2
−1
(
100 μL). The compound was purified by semipreparative RP-
HPLC (Column 6) with isocratic elution of Buffer A (0.1%
N
DOI: 10.1021/acs.inorgchem.7b01247
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
−
1
mg mL ) in a separate evacuated vial, and a 0.5 mL aliquot was
taken from both solutions and mixed together. To the solution
Paul S. Donnelly: 0000-0001-5373-0080
Funding
was added [9 TcO ] (0.1 mL in 0.9% saline, 108 MBq). The
conjugated peptide, H2L (cRGDfK) or H2L (Tyr -Oct),
was dissolved in degassed Milli-Q water (1 mg mL ), and 100
μL of this mixture was added to the technetium solution. The
sample was neutralized with NaHCO (pH 6.5, approximately
5 μL) then filtered or allowed to react without neutralizing at
ambient temperature for 30 to 120 min. The samples were
filtered (Supelco, Iso-disc Filter, 4 mm x 0.45 μm). Radio-
chemical yields were evaluated by reverse-phase high-perform-
ance liquid chromatography (Column 7, System C).
9m
−
4
Australian Research Council FT130100204 (P.S.D.). People
Programme (Marie Curie Actions) of the European Union’s
Seventh Framework Programme (FP7/2007−2013) under
REA Grant Agreement Number 299009 (MTM). The Centre
of Excellence in Medical Engineering Centre funded by the
Wellcome Trust and EPSRC (WT088641/Z/09/Z), the KCL
and UCL Comprehensive Cancer Imaging Centre funded by
CRUK and EPSRC in association with the MRC and DoH
1
−4
1−3
3
−1
3
5
(
England), and the NIHR Biomedical Research Centre at Guy’s
and St Thomas’ NHS Foundation Trust and King’s College
Stability Studies in Human Serum. Human blood samples
were centrifuged with a Heraeus Labofuge 6000 centrifuge at
000g for 10 min (Heraeus, Hanau, Germany). Radioactivity
London.
Notes
3
The authors declare no competing financial interest.
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 coef-
ficient 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).
ACKNOWLEDGMENTS
■
Professor Jonathan R. Dilworth (University of Oxford) is
acknowledged for the central role he played in the early aspects
of this research. We acknowledge the generous support of Prof.
Andrew M. Scott, A/Prof. Henri Tochon-Danguy, Dr. FT Lee,
Nick Alexopoulos, and David Thomas of the Austin Hospital,
(
9
9m
Heidelberg, Victoria, Australia, for providing access to
Tc
For serum stability studies, blood from a healthy male (20
mL) 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 were discarded. An aliquot
of plasma (0.6 mL) was added to labeled compound,
and radiochemistry facilities. This research was supported by
the Australian Research Council, FT130100204 (PSD). M.T.M.
acknowledges the support of the People Programme (Marie
Curie Actions) of the European Union’s Seventh Framework
Programme (FP7/2007-2013) under REA Grant Agreement
Number 299009. This research was supported by the Centre of
Excellence in Medical Engineering Centre funded by the
Wellcome Trust and EPSRC (WT088641/Z/09/Z), the KCL
and UCL Comprehensive Cancer Imaging Centre funded by
CRUK and EPSRC in association with the MRC and DoH
99m
3
3
+
[
Tc(HL (Tyr -Oct)) ] (0.15 mL), the radioactivity was
2
monitored and the mixture was then incubated at 37 °C.
Aliquots (0.1 mL) of the mixture were removed from heating at
1
0 min and after 2 h. Acetonitrile (0.1 mL) was added to the
serum/tracer mix to precipitate serum proteins. The suspension
was shaken for 5 min and 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 (Column 7, System C) for UV and radioactivity
analysis and the pellet were washed with acetonitrile (3 × 0.1
mL), and radioactivity levels were again recorded (radioactivity
levels of the pellet were negligible).
(
England), and by the NIHR Biomedical Research Centre at
Guy’s and St Thomas’ NHS Foundation Trust and King’s
College London. The views expressed are those of the authors
and not necessarily those of the NHS, the NIHR or the DoH.
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