Preparation of tetrazine-containing [2 + 1] complexes of 99mTc and: In vivo targeting using bioorthogonal inverse electron demand Diels-Alder chemistry

Article abstract of DOI:10.1039/c7dt01497j

The aim of this work was to synthesize and evaluate [2 + 1] ^99mTc(i) polypyridine complexes containing tetrazines, which along with the corresponding Re(i) complexes, represent a new class of isostructural nuclear and turn-on luminescent probes that can be derivatized and targeted using bioorthogonal chemistry. To this end, [2 + 1] complexes of ^99mTc(i) of the type [^99mTc(CO)₃(N^N)(L)] (N^N = bathophenanthroline disulfonate (BPS) or 2,2′-bipyridine (bipy)), where the monodentate ligand (L) was a tetrazine linked to the metal through an imidazole derivative, were prepared. The desired products were obtained in nearly quantitative radiochemical yield by adding [^99mTc(CO)₃(N^N)(OH₂)]ⁿ to the imidazole-tetrazine ligand and heating at 60 °C for 30 min. Measurement of the reaction kinetics between the tetrazine and (E)-cyclooct-4-enol revealed a second-order rate constant of 8.6 × 10³ M^-1 s^-1 at 37 °C, which is suitable for in vivo applications that require rapid coupling. Stability studies showed that the metal complexes were resistant to ligand challenge and exhibited reasonable protein binding in vitro. Biodistribution studies of the more water-soluble BPS derivative in normal mice, one hour after administration of a bisphosphonate derivative of trans-cyclooctene (TCO-BP), revealed high activity concentrations in the knee (9.3 ± 0.3 %ID g^-1) and shoulder (5.3 ± 0.7 %ID g^-1). Using the same pretargeting approach, SPECT/CT imaging showed that the [2 + 1] tetrazine complex localized to implanted skeletal tumors. This is the first report of the preparation of ^99mTc complexes of BPS and demonstration that their tetrazine derivatives can be used to prepare targeted imaging probes by employing bioorthogonal chemistry.

Full text of DOI:10.1039/c7dt01497j

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ARTICLE
Preparation of tetrazine-containing [2 + 1] complexes of 99mTc and
in vivo targeting using bioorthogonal inverse electron demand
Diels-Alder chemistry
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
b
c
c
DOI: 10.1039/x0xx00000x
Abdolreza Yazdani, Nancy Janzen, Shannon Czorny, Robert Ungard, Tanya Miladinovic, Gurmit
c
a,b
Singh, and John F. Valliant*
www.rsc.org/
The aim of this work was to synthesize and evaluate [2 + 1] 99mTc(I) polypyridine complexes containing tetrazines, which
along with the corresponding Re(I) complexes, represent a new class of isostructural nuclear and turn-on luminescent
probes that can be derivatized and targeted using bioorthogonal chemistry. To this end, [2+1] complexes of 99mTc(I) of the
type [99mTc(CO)
(N^N)(L)] (N^N = bathophenanthroline disulfonate (BPS) or 2,2'-bipyridine (bipy)), where the monodentate
3
ligand (L) was a tetrazine linked to the metal through an imidazole derivative, were prepared. The desired products were
obtained in nearly quantitative radiochemical yield by adding [99mTc(CO)
)] to the imidazole-tetrazine ligand
n
3
(N^N)(OH
2
and heating at 60 °C for 30 min. Measurement of the reaction kinetics between the tetrazine and (E)-cyclooct-4-enol
3
-1 -1
revealed a second-order rate constant of 8.6 × 10 M s at 37 °C, which is suitable for in vivo applications that require
rapid coupling. Stability studies showed that the metal complexes were resistant to ligand challenge and exhibited
reasonable protein binding in vitro. Biodistribution studies of the more water-soluble BPS derivative in normal mice, one
hour after administration of a bisphosphonate derivative of trans-cyclooctene (TCO-BP), revealed high activity
concentrations in the knee (9.3  0.3 %ID/g) and shoulder (5.3  0.7 %ID/g). Using the same pretargeting approach,
SPECT/CT imaging showed that the [2 + 1] tetrazine complex localized to implanted skeletal tumors. This is the first report
of the preparation of 99mTc complexes of BPS and demonstration that their tetrazine derivatives can be used to prepare
targeted imaging probes by employing bioorthogonal chemistry.
Of the bioorthogonal chemical reactions available, the inverse
electron demand Diels-Alder (IEDDA) cycloaddition reaction
Introduction
Bioorthogonal coupling reactions are highly selective chemical
transformations that can take place in vitro and in vivo. They are
generally insensitive to the biological surrounding and do not
between tetrazines and strained alkenes such as trans-cyclooctene
5–9
(
TCO) have proven to be particularly effective. Their success is
driven largely by rapid reaction kinetics, high selectivity,
compatibility with biological media, and the ease of synthesis of
1,2
interfere with ongoing biochemical processes.
As a result,
bioorthogonal chemistry in conjunction with pretargeting strategies
have been used to develop a wide range of different targeted
imaging probes. Here, one reaction component is coupled to a
targeting agent that is administered and allowed to localize at the
site of interest. This is followed by administration of the second
reaction component that is linked to a signaling moiety, such as a
fluorophore or a compound containing a radionuclide, which
selectively couples to the first reagent. This approach can result in
enhanced image contrast and in the case of radiotherapeutic
agents, the delivery of higher doses, compared to conventional
1
0–16
TCO and tetrazine derivatives.
A wide range of radiolabeled
11
18
tetrazines have been reported including those containing C, F,
64
89
99m
111
177
12,17–22
Cu, Zr, Tc, In, and Lu.
Recently, a [2+1] type Re(I) polypyridine complex containing a
tetrazine linked through a monodentate pyridine ligand was
reported as a phosphorogenic bioorthogonal probe. The compound
was shown to effectively undergo the IEDDA reaction and have
2
3
significant dienophile induced emission enhancement leveraging
24
the luminescent properties of the Re(I) complex, and the use of
25–32
tetrazines as quenchers to create a turn-on type optical probes.
“
actively” targeted constructs.3,4
One complex was used effectively to tag (1R,8S,9s)-
bicyclo[6.1.0]non-4-yn-9-ylmethanol derived proteins in vitro
99m
a.Department of Chemistry and Chemical Biology, McMaster University, 1280 Main
suggesting that a [2+1] tetrazine complex containing
Tc in place
St. West, Hamilton, ON L8S 4M1, Canada.
Centre for Probe Development and Commercialization, 1280 Main St. West,
Hamilton, ON L8S 4M1, Canada.
Department of Pathology and Molecular Medicine, McMaster University, 1280
of its congener Re could potentially be used to visualize targets in
vivo using radioimaging methods.
b.
c.
The second-order rate constants for the reported Re(I) tetrazine
complexes indicate the reaction kinetics are not fast enough for in
Main St. West, Hamilton ON L8S 4M1, Canada.
Email: valliant@mcmaster.ca
23
vivo applications. Consequently, we investigated the preparation
Electronic Supplementary Information (ESI) available: Compound characterization
data including 1H- and 13C-NMR, HRMS, IR spectra, and HPLC; stability study data
and in vitro and in vivo testing of a new class of tetrazine-containing
including all HPLC UV- and γ-chromatograms; reaction kinetic data; biodistribution
data; and absorption and emission spectra.
See DOI: 10.1039/x0xx00000x
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99mTc(I) [2 + 1] type complexes. The tetrazine was linked to the
metal through a monodentate imidazole ligand, where the reaction
kinetics at the tracer level and the impact of changing the bidentate
ligand on the stability and extent of protein binding were evaluated.
The ability of the lead compound to localize to areas of active bone
turnover and tumor invasion in vivo, using pretargeting and the
IEDDA reaction, was also assessed.
View Article Online
DOI: 10.1039/C7DT01497J
Results and discussion
Scheme 1 Synthesis of the imidazole-tetrazine ligand 5
+
(BPS)(ImTz)]-
Synthesis of [Re(CO)
3
(bipy)(ImTz)] and [Re(CO)
3
Recently, we reported that [2 + 1] complexes of 99mTc of the type acid linker to imidazole to form 3, which was achieved in 29% yield
M(CO) (bipy)(L)] and [M(CO)
(BPS)(L)] (bipy = 2,2'-bipyridine, BPS = over two steps (Scheme 1). Compound 3 was subsequently coupled
[
3
3
bathophenanthroline disulfonate, L = substituted-imidazoles) can to the commercially available amino tetrazine 4 to form 5 (ImTz) in
be synthesized in aqueous media in nearly quantitative yield. The 73% yield.
resulting imidazole complexes are sufficiently stable in vivo that
they can be used to prepare targeted isostructural nuclear and under the conditions typically used to prepare [2 + 1] Tc/Re
One potential concern was that the tetrazine would decompose
33
optical probes. Rather than have the tetrazine bound directly to complexes or that it could act as competing donor groups reducing
9
9mTc, or in close proximity, a spacer group was deemed necessary yields and complicating purification.
34
The Re complexes
+
-
to ensure steric or electronic influences of the metal complex would [Re(CO)
not have detrimental effects on the reaction kinetics of subsequent as reference standards for the target
3
(bipy)(ImTz)] and [Re(CO)
3
(BPS)(ImTz)] , which are needed
9
9m
Tc complexes, were
IEDDA reactions. The first step was therefore to prepare the prepared by combining 5 with 7a or 8a and heating the mixtures to
appropriate monodentate ligand containing a tetrazine and reflux in either acetone or methanol for 6 h (Scheme 2). The desired
imidazole separated by a spacer. This involved adding a pentanoic
+
(BPS)(ImTz)] 10a/b (M = Re or 99mTc). The counter ions for 8a and 8b are
-
Scheme 2 Synthesis of [M(CO)
3
(bipy)(ImTz)] 9a/b and [M(CO)
3
Na . The reaction conditions shown are those used to prepare the 99mTc complexes. The conditions used to prepare the Re complexes can
+
be found in the experimental section.
2
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products 9a and 10a were purified by column chromatography in
1
13
4
8% and 37% yield, respectively. The H NMR and C NMR data of
the products were consistent with the structures of 9a and 10a, and
the HRMS for all complexes showed the expected m/z values
100
(
Supporting Information, Figures S5-S12). The ν(CO) stretching
90
80
70
60
50
40
30
-1
-1
bands for 9a appear at 2029 and 1916 cm , and 2025 and 1906 cm
for 10a which are within the range reported for comparable
complexes containing the [Re(CO) ] core.
3
+
35
Photophysical Properties
Although the primary focus of the work was to prepare and
evaluate
emission spectra and turn-on ratio for 9a and 10a were measured.
Compounds 9a and 10a showed dominant absorption bands at 290
nm attributed to intra-ligand transitions and lower energy MLCT
9
9m
Tc complexes, for completeness, the absorption and
1
absorption bands at 330 and 365 nm respectively. In the presence
of excess TCO-OH, 9a and 10a had a 2.5 and 9-fold increase in the
emission intensity (λmax = 597 nm for 9a, and λmax = 625 nm for 10a)
respectively (Supporting Information, Figures S36, S37). These turn-
on ratios are significant but modest compared to other tetrazine-
20
1
0
0
31,36
fluorophore derivatives,
which is due to the distance of the
0
2
4
6
tetrazine from the Re-complex and its ability to freely rotate
relative to the bidentate metal complex. As the goal was to prepare
probes for radioimaging, the turn-on ratio is less relevant than
ensuring the tetrazine is unencumbered by the metal complex with
respect to its ability to couple rapidly with TCO-derived
biomolecules.
time (h)
Fig. 1 Protein binding and stability studies for 9b and 10b in mouse
plasma. Protein binding was determined by counting radioactivity in
precipitate pellet (after acetonitrile treatment and centrifugation)
and in the supernatant, using a gamma counter. The graph shows
the percentage of total Tc activity (% activity) found in the pellet
), and supernatant () for 9b, the pellet () and supernatant ()
Radiochemistry
99m
To prepare the 99mTc analogues of 9a and 10a, [99mTc(CO)
2 3
(H O)
]+ 6
3
(
was first combined with 2,2'-bipyridine (bipy) and the mixture
for 10b and the percentage (% activity) of intact 9b () and 10b ()
in supernatants as determined by radioHPLC.
99m
+
heated to 110 °C for 6 min to give [ Tc(CO)
3 2
(bipy)(H O)] 7b
(
Scheme 2). The more water soluble BPS ligand was combined with
6
[
and the mixture heated at 40 °C for 15 min to produce
99mTc(CO)
-
3
(BPS)(H
2
O)] 8b. Both intermediates were in turn
3
7 °C for 6 h.38 This is higher than the concentration of these amino
acids in the blood and is a convenient way to assess stability prior to
converted to the desired products through treatment with the ImTz
ligand 5 and heating at 60 °C for 30 min. The formation of desired
products 9b and 10b were confirmed by comparison of the HPLC
retention times to that for the Re standards (Supporting
Information, Figures S13, S24). The products could be readily
isolated in nearly quantitative yield by simple solid phase
extraction. The log P values were measured and were consistent
with the more hydrophilic nature of the BPS ligand (log P for 9b =
3
9
in vivo studies. Samples were periodically analyzed by radio-HPLC,
which showed that the metal complexes were resistant to ligand
challenge where there was no significant decomposition up to 6 h
(
Supporting Information, Figures S14-S23, and S25-S34).
The stability and tendency to bind proteins was further
evaluated by incubation of the metal complexes in plasma.
Compounds 9b and 10b were added to mouse plasma at 37 °C and
the amount of activity bound to proteins as a function of time was
determined following precipitation with acetonitrile (Figure 1). The
0
.33; log P for 10b = - 0.39).
Stability Studies
One potential concern with the use of [2 + 1] complexes to create stability of the complexes in the resulting supernatant was
targeted imaging probes is the premature loss of the monodentate determined by HPLC. The data showed increasing but modest levels
ligand in vivo. This can result in high protein binding and elevated of plasma protein binding over time reaching a maximum value of
37
background activity in the liver. In the case of the tetrazine approximately 30% at 6 h. At 1 h, more than 80% of both metal
derivatives, loss of the ligand would also preclude the complex from complexes were still intact, which based on data for other
undergoing the IEDDA reaction and localizing at sites containing radiolabeled tetrazines is sufficient to achieve efficient coupling to
TCO-derived targeting molecules. The stability of the 99mTc-tetrazine TCO-derived biomolecules in vivo.11,40 The HPLC chromatograms did
complexes was assessed in vitro by amino acid challenge and not show any evidence of loss of the monodentate ligands (i.e.
9
9m
+
plasma stability studies. For the amino acid challenge, 9b and 10b there was no evidence of the formation of [ Tc(CO)
3
(bipy)(OH
2
)]
99m
-
were incubated with 2 mM cysteine or histidine in PBS (pH = 7.4) at 7b, or
[
Tc(CO) (BPS)(OH )] 8b) where the observed
3 2
decomposition is more likely associated with the reactive tetrazine
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moiety, where comparable decomposition has been reported for by 10b. The mice were sacrificed at 6 and 12 h after injection of
View Article Online
DOI: 10.1039/C7DT01497J
10b, and radioactivity in various tissues and fluids counted.
other radiolabeled tetrazines.
Biodistribution data at
6
h
showed significant activity
Kinetics
concentrations at the sites of elevated bone metabolism including
in the knee (9.3  0.3% ID/g) and shoulder (5.3  0.7% ID/g). High
concentrations were also seen in the gall bladder (54.3  5.4% ID/g),
large intestine and caecum (27.7  4.6% ID/g) which decreased to
For use in vivo, IEDDA reactions between tetrazines and TCO-
derivatives should have second-order rate constants in excess of
3
-1 -1 41
10
M s . In order to study the reactivity of the appended
tetrazine and any potential influence of the metal complex, the rate
of reaction of a [2 + 1]
derivative was studied. [99mTc(CO)
with increasing concentrations of (E)-cyclooct-4-enol (TCO-OH). At
select times, samples were quenched by adding an excess of the
tetrazine 4 and the extent of formation of the product determined
by HPLC. The data was fitted to a first-order exponential and a
pseudo first-order rate constant (kobs) calculated. The second-order
rate constant was determined by plotting the kobs vs. the
(
2
7.9  2.8% ID/g) and (3.0  0.7% ID/g) at 12 h, respectively (Figure
). The data clearly demonstrate that 10b is sufficiently stable to be
99m
Tc-tetrazine complex with a model TCO
+
3
(bipy)(ImTz)] 9b was combined
able to ligate with the bound TCO-BP in vivo. Had the compound
lost the tetrazine ligand prior to coupling with the TCO-BP
derivative, the biodistribution results would have showed no uptake
in the bone. This is based on an imaging study performed using 8b
in the same animal model, which showed almost exclusive uptake
in the liver and no obvious uptake in the bone.
42
For the SPECT/CT imaging studies, TCO-BP was administered 1 h
before 10b and then imaging was carried out at 6 and 24 h after the
technetium complex was injected. The resulting images were
consistent with the biodistribution data and clearly showed high
uptake and long residence time at the sites of elevated bone
metabolism, especially the knee joints (Figure 3). The significant
liver, gall bladder, and intestinal uptake is due in part to the
hydrophobic tetrazine ligand, since prior work has shown that polar
derivatives of the BPS complex have significantly lower levels in
3
-1 -1
concentration of TCO-OH and was found to be 8.6 × 10 M s at 37
C (Supporting Information, Figure S38). This rate is comparable to
°
other radiolabeled tetrazines that have been used successfully in
vitro and in vivo and significantly higher than for [2+1] Re
complexes where the tetrazine was linked to the metal through
pyridyl ligands in the absence of any additional spacer group.17,23
Biodistribution Studies and SPECT Imaging
For in vivo testing, we opted to employ the more polar BPS complex
42
these tissues.
42
1
0b. Biodistribution studies were performed in female BALB/c
mice using a TCO-derivative of a bisphosphonate (alendronate), Imaging Bone Tumors
which is capable of binding sites of active bone metabolism.21
solution of the trans-cyclooctene (TCO-BP) (approximately 20
mg/kg in saline) was administered intravenously, followed 1 h later
A
Having demonstrated that 10b would undergo the IEDDA reaction
with TCO-BP in vivo, a subsequent study was performed in a mouse
bone tumor model, where lesions are known to contain active bone
70
60
50
40
30
20
10
0
6
h
1
2h
Organ/Tissue
Fig. 2 Biodistribution data for select fluids and tissues for pretargeting with 20 mg/kg of TCO-BP administered 1 h prior to 10b. Experiments
were performed using BALB/c mice (n = 3 per time point) with tissues collected at 6 h and 12 h post administration of the labeled
compound. Data are expressed as the mean percent injected dose per gram (%ID/g) ± SEM.
4
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intravenously, and 10b was injected 1 h later. For comparison,
imaging studies were also performed using the same model and
99m
Tc-MDP, which is employed clinically for imaging bone
metastases.45 Both labeled compounds showed significant
accumulation in the tumor-bearing femur (Figure 4), with seemingly
less but non-negligible accumulation in the contralateral knee joint,
which as mentioned was also observed in normal mice imaged
following administration of TCO-BP and 10b (Figure 3). A region of
interest analysis of the SPECT data comparing the tumor-bearing
and non-tumor bearing knee joints (Supporting Information, Figure
S39) supported this observation. However, determining the exact
difference between localization of 10b in the tumor-bearing and
control joints would require quantitative biodistribution study,
which is challenging in this particular model given that the tumor is
embedded in the bone.
There was a significant amount of activity observed in the
intestine and liver, which as noted previously is likely the result of
the lipophilic nature of the tetrazine complex. In contrast, the
Fig. 3 SPECT/CT images of healthy BALB/c mice administered 10b
1
2
h after TCO-BP (20 mg/kg). Images were obtained at (a) 6 and (b)
99m
4 h after administration of the
Tc complex, and were set at to
9
9m
hydrophilic
Tc-MDP compound had no significant localization to
the same threshold. Red arrows indicate SPECT signal from knees.
Other SPECT signal is evident from liver (L), intestine (I), and bladder
the liver or intestine. The goal here was not to make an agent that
99m
is superior to Tc-MDP, but rather to show that [2 + 1] complexes
bearing a tetrazine have sufficient stability and reactivity to
effectively undergo bioorthogonal coupling reactions in vivo. Having
demonstrated this for the first time, future efforts will focus on
tuning the pharmacokinetics by modifying the polarity of the
tetrazine and linker group.
(
B). Color scale bar indicates % intensity of image signal.
metabolism and can be detected using radiolabeled
4
3-45
bisphosphonates.
An established mouse syngeneic model of
breast cancer metastasis, involving injection of mouse 4T1
mammary carcinoma cells into the distal epiphysis (head) of the left
46,47
femur was utilized for the study.
TCO-BP was administered
100
L
L
50
I
I
B
B
0
Fig. 4 SPECT/CT images of BALB/c mice bearing 4T1 tumors in the femur following administration of TCO-BP 1 h prior to the 99mTc-complex
99m
99m
1
0b, or Tc-MDP alone. Images were obtained at 6 h and 24 h post administration of 10b (a, b) or Tc-MDP (e, f). The corresponding
transverse SPECT images (c,d and g,h) through the knees, spine, and bladder are also presented. Red arrows indicate the 4T1 tumors. Other
SPECT signals are evident from liver (L), intestine (I), and bladder (B). Color scale bar indicates % intensity of image signal.
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Phenomenex Gemini C-18 semipreparative column (250×10 mm, 5
µm) operating at a flow rate of 4.0 mL/min were used for all
Conclusions
In summary, [2 + 1] complexes of Tc(I) containing a tetrazine
substituent were developed and evaluated in vitro and in vivo for
the first time. The
analyses. The solvent gradient was: (solvent A = H
solvent B = CH CN + 0.1% TFA): 0-2 min 2% B, 2-20 min 100% B; 20-
2 min 100% B, 22-23 min 2% B, 23-25 min 2% B.
2
O + 0.1% TFA,
3
99m
Tc-compounds showed good stability in vitro
2
and moderate protein binding in plasma. The previously unknown
99m
Tc-BPS-tetrazine complex 10b selectively coupled to a TCO-
derived bisphosphonate in vivo at sites of active bone remodeling Synthetic Procedures
and damage in normal and tumor bearing mice. Despite the
5
-Imidazol-1-yl-pentanoic acid 4-[1,2,4,5]tetrazin-3-yl-benzylamide
tetrazine being linked to the radiometal through a monodentate
ligand, [2+1] complexes were sufficiently stable to undergo the
IEDDA reaction in vivo and for the coupled product to be retained at
the target over multiple hours.
(ImTz) 5. 4-(1,2,4,5-Tetrazin-3-yl)phenyl) methanamine hydro-
chloride (8.0 mg, 0.045 mmol) in DMF (3 mL) was added to a
premixed solution of (5-imidazole-1-yl)pentanoic acid (15 mg, 0.09
mmol), diisopropylethylamine (16 µL, 0.09 mmol), and benzotriazol-
1
-yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP)
Experimental
(
62 mg, 0.12 mmol) in DMF (3 mL). The resulting solution was
stirred overnight, the solvent evaporated, and the compound
subsequently purified by silica-gel column chromatography using
Materials and instrumentation
All solvents were purchased from Caledon (Georgetown, ON). MeOH:DCM (1:10 v/v). The pure product was isolated as a pink
1
Chemicals were purchased from Sigma-Aldrich (Burlington, ON) and solid. (11 mg, 73% yield). mp 140-142 °C. H NMR (600 MHz,
used without further purification, unless otherwise stated. CD OD): δ (ppm) = 10.26 (s, 1H); 8.51 (d, J = 8.3 Hz, 2H); 7.65 (s, 1H);
3
Compounds (5-imidazole-1-yl)pentanoic acid methyl ester (2), (5- 7.53 (d, J = 8.3 Hz, 2H); 7.14 (s, 1H); 7.05 (s, 1H); 6.94 (s, 1 H); 4.45
imidazole-1-yl)pentanoic acid (3), [Re(CO
3
)(bipy)(Cl)] 7a, and (d, J = 6.14 Hz, 2H); 4.00 (t, J = 6.95 Hz, 2H); 2.25 (m, 2H); 1.79 (m,
2-
13
[
Re(CO
methods.
performed according to literature procedures.
solvents for NMR samples were purchased from Cambridge Isotope found: 338.1719. HPLC (UV 220 nm): R = 8.4 min.
3
)(BPS)(Cl)] 8a were prepared according to literature 2H); 1.56 (m, 2H). C NMR (150 MHz, CD OD): δ (ppm) = 174.7,
3
4
8–50
Histidine and cysteine challenge studies were 167.3, 159.0, 145.7, 138.1, 131.8, 129.3, 129.2, 128.1, 120.7, 47.7,
38
Deuterated 43.4, 36.0, 31.1, 23.4. HRMS: m/z calcd for C H N O: 338.1729
1
7 20 7
t
99m
-
Laboratories (Tewksbury, MA). [ TcO
9
4
] was obtained from a
Mo/ Tc generator (Lantheus Medical Imaging, Saint-Laurent,
[
Re(CO
3
)(bipy)(ImTz)][CF
3
SO
3
] 9a. AgOTf (26 mg, 0.10 mmol) was
)(bipy)(Cl)] 7a (46 mg, 0.10 mmol) in
9
99m
added to a solution of [Re(CO
3
99m
QC) in saline (0.9% NaCl).
Tc-MDP was purchased from Isologic
acetone (15 mL), and the mixture was heated to reflux for 2 h. The
solution was filtered through Celite, and the solvent was
evaporated under vacuum. The yellow residue was re-dissolved in
(
Burlington, ON). Caution: 99mTc is radioactive and should only be
used in a licensed and appropriately shielded facility.
Nuclear magnetic resonance (NMR) spectra ( H, 13C) were recorded acetone (10 mL) and ImTz 5 (34 mg, 0.1 mmol) added and the
on a Bruker AV600 MHz spectrometer at ambient temperature. solution heated to reflux for 6 h prior to evaporation of the solvent
Microwave-assisted reactions were performed on a Biotage to dryness. The desired product was isolated by silica-gel column
Initiator 60 microwave reactor using crimp-sealed vials. Mass chromatography using MeOH:DCM (1:10 v/v) as an orange solid (44
1
-1
1
spectrometry analyses were provided by the McMaster Regional mg, 48% yield). mp 160-162 °C. IR (ν /cm ): 2029, 1916. H NMR
CO
Centre for Mass Spectrometry on an Agilent 6340 Ion Trap LC/MS (600 MHz, CD OD): δ (ppm) = 10.34 (s, 1H); 9.23 (d, J = 5.6 Hz, 2H);
3
mass spectrometer operating in electrospray ionization (ES) mode. 8.61 (d, J = 8.2 Hz, 2H); 8.52 (d, J = 8.4 Hz, 2H); 8.31 (m, 2H); 7.81
High resolution mass spectra (HRMS) were collected on a (m, 2H); 7.78 (s, 1 H); 7.54 (d, J = 8.4 Hz, 2H); 7.05 (s, 1 H); 6.57 (s, 1
Waters/Micromass Q-Tof Global Ultima spectrometer. IR spectra H); 4.48 (s, 1H); 3.92 (t, J = 7.0 Hz, 2H); 2.22 (t, J = 7.4 Hz, 2H); 1.64
1
3
were obtained on a Biorad FTS-40 FTIR spectrometer. Fluorescence (m, 2H); 1.34 (m, 2H). C NMR (150 MHz, CD OD): δ (ppm) = 197.0,
3
spectra were collected using 50 μM solutions with the Tecan 175.3, 167.6, 159.3, 157.1, 154.8, 145.7, 142.2, 141.4, 132.3, 129.9,
Infinite M1000 plate reader. High performance liquid 129.8, 129.5, 129.3, 125.9, 122.7, 48.5, 43.7, 36.0, 31.0, 23.4.
chromatography (HPLC) was performed on a Waters 1525 Binary HRMS: m/z calcd for C H N O Re: 764.1744 found: 764.1722.
30
27
9
4
(
Midford, MA) monitored simultaneously with 2998 Photodiode HPLC (UV 254 nm): R = 15.1 min.
t
Array Detector at 220/254 nm and in-line radioactivity Bioscan
gamma detector with NaI(Tl) scintillator using the Empower
software package. Phenomenex Gemini C-18 analytical column (250
[
Re(CO
3
)(BPS)(ImTz)]- 10a. [Re(CO) (BPS)(Cl)]2- 8a (80 mg, 0.1
3
mmol) and silver triflate (26 mg, 0.1 mmol) were dissolved in MeOH
(
12 mL) and heated to reflux for 2 h. The solution was filtered
×
4.60 mm, 5 µm) operating at a flow rate of 1.0 mL/min, and
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 6
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ARTICLE
through Celite, and ImTz 5 (68 mg, 0.2 mmol) added to the filtrate. supernatant was separated from the pellet and the aVciteiwviAtryticilne Oenalicnhe
The mixture was heated to reflux for 6 h, then evaporated to was measured using a dose calibrator. ForDaOllI:t1i0m.1e03p9o/Cin7tDsTe0x1c4e9p7Jt
dryness under vacuum. The desired product was isolated by silica- t=0, pellets were washed with 100 µL of ice cold PBS and the
gel column chromatography using MeOH:DCM (1:3 v/v) as an percentage bound to blood proteins calculated as: %bound =
orange solid (46 mg, 37% yield). mp 230-234 °C. mp 241-243 °C. IR [(amount of activity in the wash pellet)]/[(amount of activity in
-1
1
(
ν
CO/cm ): 2025, 1906. H NMR (600 MHz, CD
3
OD): δ (ppm) = 10.30 pellet) +
(amount of activity in supernatant)] × 100. Values are
(
(
6
2
(
1
1
4
1
s, 1H); 9.50 (t, J = 4.4 Hz, 2H); 8.50 (d, J = 8.3 Hz, 2H); 8.17 – 8.01 reported as an average of two experiments ± standard deviation.
m, 8H); 7.80 – 7.67 (m, 5H); 7.52 (d, J = 8.3 Hz, 2H); 7.13 (s, 1H);
Kinetics. 99mTc(CO) (bipy)ImTz+ 9b was combined with increasing
3
.96 (s, 1H); 4.48 (s, 2H); 4.05 (t, J = 6.9 Hz, 2H); 2.32 (t, J = 7.3 Hz,
H); 1.82 (m, 2H); 1.64 (m, 2H). C NMR (150 MHz, CD OD): δ
3
ppm) = 198.5, 190.5, 175.6, 167.6, 159.2, 154.4, 154.3, 152.0,
48.8, 147.8, 147.5, 145.6, 138.9, 138.3, 137.3, 132.7, 132.3, 131.0,
30.4, 130.2, 129.4, 129.3, 128.3, 128.3, 127.8, 127.7, 126.9, 120.7,
concentration of TCO-OH (3, 4, and 5 µM) in 200 µL saline for 5 min.
At the selected times (15, 30, 45, 60, 120, 180, and 300 s), a 20 µL
sample was taken and quenched by the addition of 5 (1.5 µL, 5
mg/mL in DMF), and analyzed by HPLC. The data was fitted to a first
order exponential to determine the pseudo first order rate constant
13
35 9 2
7.7, 43.8, 36.3, 31.6, 23.9. HRMS: m/z calcd for C44H N O10ReS :
(kobs). A plot of kobs vs. TCO concentration was used to determine
098.1444 found: 1098.1478. HPLC (UV 254 nm): R = 14.7 min.
t
1
1
the second-order rate constant.
General Procedure for the Preparation of 7b-10b. Sodium
boranocarbonate (10 mg, 0.10 mmol), sodium carbonate (15 mg,
Cell Culture
Mycoplasma free 4T1 cells (ATCC, Manassas, VA) were maintained
at sub-confluent densities in a humidified incubator with 5% CO in
room air at 37 °C using high glucose RPMI (Life Technologies,
Carlsbad, CA) supplemented with 10% fetal bovine serum and
antibiotics (100 U/mL penicillin sodium and 100 µg/mL
streptomycin sulfate) (Invitrogen).
0
.14 mmol), sodium borate (20 mg, 0.05 mmol) and sodium
potassium tartrate (22 mg, 0.08 mmol) were placed in a microwave
vial and purged with argon for 10 min. To this mixture, Na99mTcO
1 mL, 370-740 MBq) was added and the vial was heated in a
microwave reactor at 110 °C for 3.5 min to form
2
4

(
9
9m
+

3 2 3
Tc(CO) (H O)  . The solution was cooled to RT and adjusted to
pH 5.5-6.5 with 1.0 M HCl(aq). Bipyridine (1 mg, 6 µmol) was then Animal Care
added to the solution in a new microwave vial that had been
Female BALB/c immunocompetent mice (Charles River
purged with argon, and the mixture heated to 110 C for 6 min to
Laboratories, St Constant, QC) at 4–6 weeks of age were sterile
housed and maintained at 24 °C with a 12-h light/dark cycle and
were provided autoclaved food and water ad libitum. All
procedures were conducted according to the guidelines of the
Committee for Research and Ethical Issues of the International
Association for the Study of Pain, and guidelines established by the
Canadian Council on Animal Care and the McMaster University
Animal Research Ethics Board.
99m
3 2
form  Tc(CO) (bipy)(H O)]Cl. In case of the BPS derivative, BPS (1
mg, 2 µmol) was added as a solid and the reaction mixture was
heated to 40 C for 15 min to form 99mTc(CO)
O)] . The 7b
-
3
(BPS)(H
2
and 8b solutions were subsequently added to the monodentate
ligand 5 (1 mg) under argon and the mixture stirred at 60 C for 30
min. The formation of products was confirmed by analytical HPLC.
99m
+
3 2 3
Conversion of  Tc(CO) (H O)  to the final product was > 99% for
both products.
Biodistribution studies
Purification of 99mTc Complexes
Biodistribution studies were performed using female Balb/c mice
Purification of the 99mTc complexes was achieved by solid phase
extraction. The reaction mixture was diluted with water (2 mL) and
the solution loaded on a Waters C18 Sep-Pak Plus cartridge, which
(
Charles River) at the indicated time points. The mice were
administered all compounds via tail vein injection with final volume
not exceeding 200 µL. A 5 mg/mL solution of TCO-BP in saline was
administered at a dose of 20 mg/kg. One hour after the TCO-BP
injection, the mice were administered approximately 0.2 MBq of
had been previously activated with EtOH (6 mL) and H
After loading the reaction mixture, the cartridge was washed with
5% CH CN in H O (12 mL), followed by 25% CH CN in 0.4% (w/v)
2
O (6 mL).
1
3
2
3
10b. At 6 h and 12 h post-injection of 10b, animals were
aqueous ammonium formate (6 mL). The desired products were
eluted with a 1:1 v/v mixture of 0.4% aqueous ammonium formate
and CH CN. For biological studies, the product was was evaporated
3
to dryness and reconstituted in saline as the sodium salts to the
desired activity concentration.
anesthetized with 3% isoflurane and euthanized by cervical
dislocation. Fluids, bone (knee, shoulder), and select tissues were
collected, weighed, and counted in a PerkinElmer Wizard 1470
Automatic Gamma Counter. Decay correction was used to
normalize organ activity measurements to time of dose preparation
for data calculations. Data was expressed as percent injected dose
per gram (%ID/g) or percent injected dose per organ (%ID/o).
Compound Testing and Evaluation
Plasma Binding. 99mTc complexes 9b or 10b (100 µL, 111 MBq) were
added to pre-warmed (37 C) mouse plasma (900 µL) (Innovative Tumor inoculation
Research, Novi, MI), and the mixture vortexed prior to incubation at
On day 0, mice were anaesthetized by isoflurane inhalation and
injected subcutaneously with buprenorphine (0.05 mg/kg)
37 C. At multiple time points (t=0, 0.25, 0.5, 1, 2, 3, 6 h), 100 µL
was removed and added to 200 µL ice cold CH CN. Samples were
3
(
Schering-Plough, Hertfordshire, UK) to reduce discomfort due to
vortexed and then centrifuged at maximum speed for 10 min. The
amount of activity in the whole sample was measured using a CRC-
5
the surgical procedure. Animals were inoculated with 2×10 4T1
cells in 25 μL sterile phosphate-buffered saline percutaneously into
the left distal femur. To minimize tissue damage, mice were laid
2
5R dose calibrator (Capintec Inc, Florham Park, NJ). The
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
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Journal Name
supine with the ipsilateral stifle joint bent at 90° to provide
clearance of the patella. A 26-gauge needle was then manually
inserted between the medial and lateral condyles of the distal
epiphysis parallel to the longitudinal axis of the femur to penetrate
the cortical bone and enter the epiphysis. The inoculate was infused
slowly over 1 minute. The contralateral hind limbs served as a
negative control specific to each animal. This method of
intrafemoral injection results in little damage to the surrounding
5
6
M. L. Blackman, M. Royzen and J. M. Fox, J. Am. Chem. Soc.,
View Article Online
2008, 130, 13518–13519.
DOI: 10.1039/C7DT01497J
C. Denk, D. Svatunek, T. Filip, T. Wanek, D. Lumpi, J. Fröhlich, C.
Kuntner and H. Mikula, Angew. Chem. Int. Ed., 2014, 53, 9655–
9659.
C. Denk, D. Svatunek, S. Mairinger, J. Stanek, T. Filip, D.
Matscheko, C. Kuntner, T. Wanek and H. Mikula, Bioconjugate
Chem., 2016, 27, 1707–1712.
B. Nichols, Z. Qin, J. Yang, D. R. Vera and N. K. Devaraj, Chem.
Commun., 2014, 50, 5215–5217.
M. F. Garcίa, X. Zhang, M. Shah, J. Newton-Northup, P. Cabral,
H. Cerecetto and T. Quinn, Bioorg. Med. Chem., 2016, 24, 1209–
7
5
1
tissues. Imaging studies were performed 12 days after inoculation.
8
9
Imaging
A 5 mg/mL solution of TCO-BP in saline (20 mg/kg) 1 h prior to 10b
9
9m
or a solution of
vein injection. Prior to imaging, mice were anaesthetized with 1.5-
.0% isoflurane and maintained under same conditions for the
length of the SPECT and CT scans. Imaging was conducted at 6 and
Tc-MDP (~35 MBq) were administered via tail
1215.
10 N. K. Devaraj, R. Upadhyay, J. B. Haun, S. A. Hilderbrand and R.
Weissleder, Angew. Chem. Int. Ed. 2009, 48, 7013–7016.
2
11 R. Rossin, P. R. Verkerk, S. M. van den Bosch, R. C. M. Vulders, I.
Verel, J. Lub and M. S. Robillard, Angew. Chem. Int. Ed., 2010,
99m
2
4 h post injection for 10b, and a 4 and 6 h for Tc-MDP. At each
time point, imaging was conducted on a GammaMedica Ideas X-
SPECT system (North Ridge, California). CT acquisition consisted of
49, 3375–3378.
12 B. M. Zeglis, K. K. Sevak, T. Reiner, P. Mohindra, S. D. Carlin, P.
5
12 projections acquired over 360 with 75 Kvp, 205 mA cone
Zanzonico, R. Weissleder and J. S. Lewis, J. Nucl. Med., 2013, 54,
beam CT system. Cobra Exxim software (Feldkamp filtered back-
projection cone beam reconstruction software) was used to
reconstruct the images at a voxel size of 155 microns and a matrix
1389–1396.
1
3 J. L. Seitchik, J. C. Peeler, M. T. Taylor, M. L. Blackman, T. W.
Rhoads, R. B. Cooley, C. Refakis, J. M. Fox and R. A. Mehl, J. Am.
Chem. Soc., 2012, 134, 2898–2901.
3
size of 512 . An OS-EM interactive reconstructed method (2
iterations/8 subsets) was used to reconstruct the SPECT data which
was fused to the CT data using in house software. AMIDE software
was used to analyze all images. Image analysis was performed using
Amira 3D software. From the CT images, regions of interest (ROIs)
were defined for each knee joint at 24 h, for control (no tumor) and
tumor-bearing mice, and an ROI analysis completed for the
corresponding SPECT data. The data was normalized from the
calculated ROI values, by dividing the mean value of voxels within
the ROI (signal) by the number of fractional voxels in the ROI. The
ratio of normalized signal in the tumor-bearing knee to the non-
tumor bearing knee was then calculated.
1
1
1
1
1
4 D. S. Liu, A. Tangpeerachaikul, R. Selvaraj, M. T. Taylor, J. M. Fox
and A. Y. Ting, J. Am. Chem. Soc., 2012, 134, 792–795.
5 R. M. Versteegen, R. Rossin, W. Ten Hoeve, H. M. Janssen and
M. S. Robillard, Angew. Chem. Int. Ed., 2013, 52, 14112–14116.
6 D. M. Patterson, L. A. Nazarova and J. A. Prescher, ACS Chem.
Biol., 2014, 9, 592–605.
7 R. Rossin and M. S. Robillard, Curr. Opin. Chem. Biol., 2014, 21C,
161–169.
8 M. M. Herth, V. L. Andersen and S. Lehel, J. Madsen, G. M.
Knudsen, J. L. Kristensen, Chem. Commun., 2013, 49, 3805–
3807.
1
9 Z. Li, H. Cai, M. Hassink, M. L. Blackman, R. C. D. Brown, P. S.
Conti and J. M. Fox, Chem. Commun., 2010, 46, 8043–8045.
0 B. M. Zeglis, P. Mohindra, G. I. Weissmann, V. Divilov, S. A.
Hilderbrand, R. Weissleder and J. S. Lewis, Bioconjugate Chem.,
2011, 22, 2048–2059.
Acknowledgements
The authors gratefully acknowledge funding support from the
Natural Science and Engineering Research Council (NSERC,
2
#227514) of Canada, Canadian Cancer Society (CCS, #703857), 21 A. Yazdani, H. Bilton, A. Vito, A. R. Genady, S. M. Rathmann, Z.
and the Ontario Institute for Cancer Research (OICR,
Ahmad, N. Janzen, S. Czorny, B. M. Zeglis, L. Francesconi and J.
#C.SI.015.8). The authors acknowledge the contributions of
F. Valliant, J. Med. Chem., 2016, 59, 9381–9389.
Dr. Denis Snider, who provided scientific proofreading and 22 R. Rossin, S. M. Van Den Bosch, W. Ten Hoeve, M. Carvelli, R. M.
editing in the preparation of this manuscript.
Versteegen, J. Lub and M. S. Robillard, Bioconjugate Chem.,
013, 24, 1210–1217
2
2
2
2
2
2
3 A. W.-T. Choi, K. K.-S. Tso, V. M.-W. Yim, H.-W. Liu and K. K.-W.
Lo, Chem. Commun. 2015, 51, 3442–3445.
4 K. K.-W. Lo, A. W.-T. Choi and W. H.-T. Law, Dalton Trans., 2012,
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DOI: 10.1039/C7DT01497J
TOC Graphic
99m
A new Tc-labelled tetrazine for targeted imaging using bioorthogonal chemistry was developed and
evaluated in vivo using a trans-cyclooctene derived bisphosphonate targeting regions of high bone
turnover and bone lesions.