Synthesis, radiolabelling, and biodistribution studies of triazole derivatives for targeting melanoma

Article abstract of DOI:10.1139/cjc-2016-0239

Molecular probes that target specific markers expressed in solid tumours are in demand for cancer imaging and radionuclide therapy applications. The synthesis, characterization, and in vivo evaluation of radioiodinated triazoles designed as probes to targ

Full text of DOI:10.1139/cjc-2016-0239

773
ARTICLE
Synthesis, radiolabelling, and biodistribution studies of triazole
derivatives for targeting melanoma
Stephanie M. Rathmann, Nancy Janzen, and John F. Valliant
Abstract: Molecular probes that target specific markers expressed in solid tumours are in demand for cancer imaging and radionu-
clide therapy applications. The synthesis, characterization, and in vivo evaluation of radioiodinated triazoles designed as probes to
target melanoma are described here. Compounds were prepared using a thermal click reaction between ethynylstannane and methyl
2-azidoacetate, resulting in preferential formation of the corresponding 1,4-tin triazole. The primary amine of various targeting
vectors was then coupled to the resulting tin triazole methyl ester. These precursors were labelled with no carrier added 123I or 125I and
purified by high performance liquid chromatography to give isolated radiochemical yields between 6% and 51% and radiochemical
purities of >95% in all cases. Among the evaluated compounds, N-(2-diethylamino-ethyl)-2-(4-iodo-[1,2,3]triazol-1-yl)acetamide (7a) and
N-(1-benzylpiperidin-4-yl)-2-(4-iodo-1H-1,2,3-triazol-1-yl)acetamide (7d) showed the most promising in vivo data, and their 123I-labelled
forms were used in single photon emission computed tomography computed tomography (SPECT–CT) imaging studies. The imaging
data showed excellent tumour visualization with a very high signal to noise ratio.
Key words: radioiodine, melanoma, imaging, SPECT, triazole, radionuclide therapy.
Résumé : Les sondes moléculaires ciblant des marqueurs spécifiques exprimés dans les tumeurs solides sont des composés recher-
chés dans le domaine de la cancérologie, dans des applications en imagerie et des applications thérapeutiques a` base de radionuclé-
ides. Dans le présent article, nous décrivons la synthèse, la caractérisation et l’évaluation in vivo de triazoles marqués a` l’iode radioactif
et conçus pour agir comme sondes ciblant le mélanome. Nous avons préparé ces composés a` l’aide d’une réaction thermique de type
« clic » entre l’éthynylstannane et le 2-azidoacétate de méthyle qui produit de manière préférentielle le 1,4-stannyltriazole. Nous avons
ensuite couplé l’amine primaire de divers vecteurs de ciblage a` l’ester méthylique du stannyltriazole résultant. Nous avons marqué ces
précurseurs a` l’iode radioactif (123I ou 125I) sans entraîneur ajouté, puis nous les avons purifiés par chromatographie liquide a` haute
performance afin d’isoler les composés radioactifs correspondants, que nous avons respectivement obtenus en rendement de 6 % et
de 51 %; dans tous les cas, la pureté radiochimique a été supérieure a` 95 %. Parmi les composés évalués, le N-(2-diéthylaminoéthyl)-
2-(4-iodo-[1,2,3]triazol-1-yl)acétamide (7a) et le N-(1-benzylpipéridin-4-yl)-2-(4-iodo-1H-1,2,3-triazol-1-yl)acétamide (7d) ont présenté les
résultats les plus prometteurs dans les épreuves in vivo. Ils ont par ailleurs été employés dans des études d’imagerie par tomographie
d’émission monophotonique sensibilisée par la tomodensitométrie (TEMP–TDM) sous leur forme radiomarquée a` l’iode 123 (123I). Les
données d’imagerie présentent une excellente visibilité des tumeurs et un rapport signal-bruit très élevé. [Traduit par la Rédaction]
Mots-clés : iode radioactif, mélanome, imagerie, TEMP, triazole, traitement par radionucléide.
ical target. Melanin is present in tumour lesions in over 90% of
Introduction
melanoma cases9 and can be targeted using small molecule
Melanoma accounts for 4% of all cases of skin cancer and is
benzamides that contain a pendent tertiary amine.10,11 Potential
melanin-targeted RT agents have been reported, the majority of
which are developed around an iodinated benzamide core, most
notably N-(2-diethylaminoethyl) 4-iodobenzamide (BZA) (Fig. 1).12,13
Iodine radionuclides are frequently chosen, because they allow
the production of isostructural agents for both diagnosis and ther-
apy, simply by changing the isotope used to prepare the agent.14
Radioiodinated benzamides show high tumour uptake; however,
the probes also demonstrate high nonspecific binding in vivo due
to the lipophilicity of the iodophenyl group.15,16 Changing the
prosthetic group from iodophenyl to polar heterocycles has the
potential to improve the target to nontarget uptake of a probe by
increasing hydrophilicity and decreasing retention in nontarget
tissue.17,18 By reducing nonspecific binding, it is possible to de-
velop a targeted RT agent with low radiotoxicity and that provides
a maximum dose to the tissue of interest.
responsible for 80% of all skin cancer deaths.1 Until recently, there
has been a limited number of effective treatment options for
patients with malignant melanoma. Inhibitors of the B-Raf kinase
have shown some improved results over conventional therapies
but are effective only with types of melanoma that have the spe-
cific BRAF gene mutation.2,3 In addition, tumour resistance to
these drugs and the resulting recurrence of disease are frequent.4,5
More recently, exciting clinical trial results have been obtained
with novel immunotherapies against melanoma, notably CTLA–4
and PD–1 targeting antibodies.6–8 Although these antibody thera-
pies appear to be effective, they often work for specific subpopu-
lations of patients, and long-term overall survival may require
repeat treatments or combination therapies with other antican-
cer agents. There remains a need for new therapeutics to treat late
stage and recurrent metastatic melanoma.
One option that has been explored for treating metastatic mel-
anoma is radionuclide therapy (RT) using melanin as the biochem-
Received 16 May 2016. Accepted 28 June 2016.
S.M. Rathmann, N. Janzen, and J.F. Valliant. Department of Chemistry and Chemical Biology, McMaster University, 1280 Main St. W., Hamilton,
ON L8S 4M1, Canada.
Corresponding author: John F. Valliant (email: valliant@mcmaster.ca).
Copyright remains with the author(s) or their institution(s). Permission for reuse (free in most cases) can be obtained from RightsLink.
Can. J. Chem. 00: 773–780 (0000) dx.doi.org/10.1139/cjc-2016-0239
Published at www.nrcresearchpress.com/cjc on 4 July 2016.

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Can. J. Chem. Vol. 00, 0000
Fig. 1. Comparison of the structures of iodinated benzamide (BZA) and 7a, the iodinated TAAG analogue.
Scheme 1. Synthesis of compounds 6a–6c and 7a–7c.
The Triazole Appending Agent (TAAG) is a new iodine-containing
synthon that was designed as a hydrophilic prosthetic group that
can be used in place of the commonly employed iodophenyl
group.19 TAAG, when iodinated, clears rapidly via the kidneys and
shows low nonspecific binding in vivo. The first TAAG conjugate
synthesized was a derivative designed to bind prostate specific
membrane antigen (PSMA),19 a protein overexpressed in prostate
cancer cells. This compound, when iodinated, showed high up-
take in prostate cancer tumours and low nonspecific binding to
nontarget tissues, making the TAAG group an attractive building
block for preparing a melanin-targeted imaging probe and radio-
therapeutic agent.19
A method was developed to prepare analogues of BZA, which
contained tin-triazoles as precursors for oxidative radiohalogenation
reactions. Following radiolabelling, the products were compared
with nonradioactive reference standards, and the impacts of dif-
ferent substituents on their localization in melanin-expressing
tumours and nontarget tissues were assessed in vivo using a syn-
geneic tumour model. An iterative approach was applied using
modifications to the core structure based on biodistribution re-
sults for each successive derivative to determine the functional
groups required for optimal target binding and clearance. Single
photon emission computed tomography – computed tomography
(SPECT–CT) imaging studies were then performed on the lead
compounds.
showed characteristic singlet peaks at 8.08 and 5.24 ppm, repre-
senting the triazole and adjacent methylene protons, respectively
(see Supplementary material). A multiplet at 1.13 ppm indicated
the terminal CH3 group on the targeting vector, further confirm-
ing that the coupling reaction was successful. The fluorous tin
derivatives were prepared initially to enable purification of the
radiolabelled compound by fluorous solid phase extraction.20 In
select cases, precursors were developed containing tributyl tin
derivatives (Scheme 2) in place of the fluorous groups to address
solubility issues. Here, the tributyl tin methyl ester (3) was synthe-
sized through the thermal click reaction of tributyl tin acetylene
with methyl 2-azidoacetate following a literature procedure.19
Compound 3 was subsequently coupled to different amines, pre-
pared via simple alkylation of Boc-protected 4-amino-piperidine,
in methanol at 60 °C to produce 6d–6i in 7%–64% yield (Scheme 2).
Radiochemistry
Each of the tin-triazoles (6a–6i) were initially labelled with non-
radioactive iodine to generate the HPLC reference standards re-
quired to validate the identity of the radioiodinated compounds.
A crystal structure of 7d (N-(1-benzylpiperidin-4-yl)-2-(4-iodo-1H-1,2,3-
triazol-1-yl)acetamide) was obtained (Fig. 2), which showed that
the major product was the 1,4-isomer, in agreement with the NMR
data (see Supplementary material).
The tributyl tin derivatives were radiolabelled using an
iododestannylation reaction with iodogen as the oxidant.21 The
fluorous compounds 6a–6c were labelled in methanol, whereas
reactions with compounds 6d–6i were performed using a biphasic
chloroform–water mixture. Reactions were complete within 10 min,
and the products isolated by HPLC in radiochemical yields (RCY)
between 6% and 51% (Table 1), with radiochemical purity >95% in all
cases.
Results and discussion
Synthesis of TAAG derivatives and iodinated nonradioactive
reference standards
A series of TAAG derivatives were prepared as analogues of the
previously reported iodinated BZA (Fig. 1).15 As the first step to-
wards creating the radiolabelling precursor compounds, the fluo-
rous tin methyl esters 1 and 2 were synthesized by combining the
appropriate perfluorinated tin alkyne and methyl 2-azidoacetate
via a thermal click reaction. Both the 1,4- and 1,5-isomers were
present, with the former isolated by column chromatography.19
The two resulting esters were then coupled to N,N-diethylethylene
diamine to produce 6a–6b (Scheme 1). For example, ester 1 was
coupled with N,N-diethylethylene diamine in methanol at 60 °C to
give 6a in 88% yield. The identity of 6a was confirmed through
mass spectrometry, HPLC, and NMR, where the 1H NMR spectrum
Biodistribution studies
Biodistribution studies were performed for [123/125I]–7a–7i in com-
parison with [125I]–BZA in C57Bl/6 mice bearing melanin-expressing
B16F1 tumours. Tissues, organs, and fluids were collected and
weighed 24 h after compound injection, and radioactivity was mea-
sured for each. The percent injected dosage per gram (%ID/g) of se-
lected tissues or fluids are shown in Figs. 3 and 4, and key tumour to
tissue ratios were calculated (Table 2). It is important to note that
Published by NRC Research Press

Rathmann et al.
775
Scheme 2. Synthesis of compounds 6d–6i and 7d–7i.
vivo.22,23 Compound 7b was synthesized by adding an aromatic
group in the 5 position on the triazole to enhance ␲ stacking
interactions with melanin.22 The biodistribution study of [125I]–7b
showed a 10-fold increase in tumour uptake compared with
[
[
Fig. 2. ORTEP drawing of 7d. The thermal ellipsoid probability was
50%. Hydrogen atoms are omitted for clarity.
123I]–7a (Fig. 3). However, the ideal clearance that was seen with
123I]–7a was somewhat compromised in [125I]–7b, which showed
higher but not unreasonable (2.42 vs. 0.004 %ID/g) accumulation
in the liver.
In light of the results with [125I]–7b, compound [125I]–7c was
synthesized as a derivative with intermediate polarity relative to
[
123I]–7a and [125I]–7b. The biodistribution of [125I]–7c showed tu-
mour and eye uptake of 0.77 and 3.17 %ID/g, respectively, at 24 h
(Fig. 3). Tumour uptake was eight-fold higher for [125I]–7c com-
pared with [123I]–7a. At the same time, [125I]–7c displayed ideal
clearance with tumour to liver and tumour to blood ratios of 6:1
and 15:1, respectively, although accumulation in the thyroid was
somewhat elevated. Thyroid localization in biodistribution stud-
ies is indicative of metabolic release of iodide. Compound 7d was
then synthesized to combine the structural features that pro-
duced the positive results from 7b and 7c, having both the piper-
idine and phenyl rings within the molecule. The biodistribution
study of [125I]–7d revealed tumour and eye uptake of 2.21 and
6.59 %ID/g, respectively, at 24 h, which were the highest values
among all tested compounds (Fig. 3; Supplementary material).
Table 1. Log P and radiochemical
yield (RCY) values for compounds
[
123I]–7a and [125I]–7b–7i.
Compound Log P7.4
BZA 1.34
RCY (%)
42
[
[
[
[
[
[
[
[
[
123I]–7a
125I]–7b
125I]–7c
125I]–7d
125I]–7e
125I]–7f
125I]–7g
125I]–7h
125I]–7i
–0.64 0.08 18
0.63 0.05 19
–0.12 0.02 33
[
125I]–7d had 10-fold lower accumulation in the liver compared
0.70 0.19
1.02 0.37
1.33 0.27
1.14 0.19
1.39 0.25
0.84 0.11
51
6
13
40
38
27
with [125I]–BZA.
The results from these initial biodistribution studies showed
that the clearance of this new class of compounds is not solely
dependent on lipophilicity. For instance, the log P values for
[
125I]–7b and [125I]–7d are 0.63 and 0.70, respectively (Table 1). How-
Note: Log P data for BZA were from
previously reported work.23
ever, despite having similar log P values, the in vivo clearance of
these probes was quite different. The tumour to liver ratios were
0.37:1 and 27:1 for [125I]–7b and [125I]–7d, respectively, which is a
large and significant difference for probes of similar polarity. This
observation suggested that a mechanism other than the lipophi-
licity is guiding clearance and that retaining the 1,4 triazole core
was needed to promote clearance from nontarget tissues.
melanin binding can be assessed by evaluating the extent of local-
ization to the tumour and the eyes; both of which express the
target.15
The first compound tested was [123I]–7a, the TAAG analogue of
BZA. [123I]–7a showed low uptake in nontarget tissues, as well as
low (<0.1 %ID/g) tumour uptake at 24 h. However, uptake in the
eyes was 1.50 %ID/g, suggesting that [123I]–7a did indeed target
melanin (Fig. 3). It is also important to note that although the
tumour to blood ratio of [123I]–7a was less than that of [125I]–BZA
(19:1 versus 183:1, respectively), [123I]–7a had a superior tumour to
liver ratio compared with [125I]–BZA (97:1 versus 21:1, respectively).
This result supports the hypothesis that nonspecific binding can
be decreased through the use of an iodinated heterocycle in place
of the iodo-aryl prosthetic group.
Although [125I]–7d showed promising biodistribution results,
higher uptake in the tumour might be necessary for use as a
radiotherapeutic agent. Therefore, further derivatization was ex-
plored to enhance tumour uptake. We first attempted to alter the
electron density of the phenyl group through the use of electron-
donating and electron-withdrawing substituents. The presence of
the electron-donating groups was expected to increase electron
density in the ring and, therefore, provide a stronger interaction
with melanin, which is a known ␲ electron acceptor.22 The first
derivative, [125I]–7e, contained two methoxy groups and had a
tumour and eye uptake of 1.32 and 7.36 %ID/g, respectively, at 24 h
(Fig. 4). The second derivative, [125I]–7f, contained two electron-
withdrawing nitro groups and had a tumour and eye uptake of
In an attempt to improve tumour uptake, substituents on the
tertiary amine and triazole were varied because altering the size
of the amine and arene are known to impact melanin binding in
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Can. J. Chem. Vol. 00, 0000
Fig. 3. Biodistribution data (%ID/g) for selected tissues and fluids comparing radioiodinated BZA with radioiodinated compounds 7a–7d at 24 h
postinjection. Error bars are the standard errors of the mean (n = 3–5). Tissues and fluids are indicated on the x axis. Complete biodistribution data
can be found in the Supplementary material.
Fig. 4. Biodistribution data (%ID/g) for selected tissues and fluids comparing radioiodinated BZA with radioiodinated compounds 7d–7i at 24 h
postinjection. Error bars are the standard errors of the mean (n = 3). Tissues and fluids are indicated on the x axis. Complete biodistribution data can
be found in the Supplementary material.
Table 2. Tumour to blood and tumour to tissue ratios
of [123/125I]–TAAG derivatives from biodistribution data,
using C57BL/6 mice bearing B16F1 tumours (24 h post-
injection).
1.15 and 8.06 %ID/g, respectively, at 24 h. These results show that
the addition of electron-donating and electron-withdrawing sub-
stituents are overall deleterious to tumour and eye uptake, which
was unexpected, as the increased electron density should enhance
the interaction with melanin. Steric hindrance about the phenyl
ring may be the cause of the decreased uptake.
Ratios
Compound Tumour:blood Tumour:liver Tumour:eyes
A series of the fluorinated compounds (7 g–7i) showed a some-
what lower tumour uptake at 24 h compared with the parent
compound [125I]–7d (Fig. 4). Although the 24 h tumour uptake of
fluorinated [125I]–7h and [125I]–7i were slightly lower than [125I]–7d,
they retained the excellent tumour to nontumour ratios seen with
many of these TAAG constructs.24 Interestingly, the fluorine in
the para position of [125I]–7i resulted in lower thyroid localization
than did [125I]–7d (2.2 vs. 7.7 %ID/g, respectively). Compared with
BZA
162.5
19.0
89.0
15.0
221.0
66.0
115.0
103.0
189.0
191.0
6.6
9.0
0.4
0.5
0.1
0.1
0.2
0.3
0.2
0.1
0.2
0.4
0.2
[
[
[
[
[
[
[
[
[
123I]–7a
125I]–7b
125I]–7c
125I]–7d
125I]–7e
125I]–7f
125I]–7g
125I]–7h
125I]–7i
6.4
27.6
10.2
8.2
11.4
63.0
17.4
[
125I]–BZA, the equivalent TAAG derivative [123I]–7a had similar
thyroid uptake (Fig. 3), whereas uptakes of [125I]–7e and [125I]–7f
were somewhat higher (Fig. 4). Overall, when compared with the
established benzamide [125I]–BZA agent, TAAG derivatives [123I]–7a
Published by NRC Research Press

Rathmann et al.
777
Fig. 5. SPECT–CT images of mice given either (A) [123I]–7a or (B) [123I]–7d at 24 h postinjection. Tumours are indicated by yellow arrows. For
[
123I]–7d, uptake in the eyes is indicated by the white arrow. Note that images are presented at different intensities. To compare relative
uptake, refer to the corresponding biodistribution data in the Supplementary material. [Colour online.]
and [125I]–7d had lower tumour uptake but had greatly reduced
uptake by nontarget tissues (Fig. 4; Supplementary material).
230 instrument, fitted with a Varian ProStar model 330 PDA de-
tector, an IN/US ␥-RAM gamma detector, and a Star 800 analog
interface module, as well as a Waters 2489 HPLC equipped with a
Waters 2489 UV/Vis (␭ = 254 nm) and (or) a Bioscan gamma detector
(model 106). The radiolabelled compounds were isolated and ana-
lyzed using a C18 column (Waters X-Bridge 5 ␮m, 4.6 mm × 100 mm).
Samples were eluted in HPLC-grade water containing 0.1% TFA
(eluent A) for 3 min, followed by gradient increase in acetonitrile
containing 0.1% TFA (eluent B), from 10% to 100% over 13 min, at
1 mL/min.
SiliaFlash P60 silica gel from SiliCycle (Québec City, QC) was
used for silica gel chromatography. Toluene and tetrahydrofuran
were distilled using a PURE SOLV distillation system. FC–72 was
purchased from 3M Canada (London, ON), and (CF3(CF2)5(CH2)2)3SnPh
was purchased from Fluorous Technologies (Ambridge, PA). All
other reagents were purchased from Sigma-Aldrich (Oakville, ON)
and AK Scientific (Union City, CA) and used without further puri-
fication. 123I was produced by MDS Nordion (Vancouver, BC) using
the 124Xe(p, 2n) reaction and was delivered as a [123I]Na in 0.1 mol/L
NaOH solution. 125I was produced by the McMaster Nuclear Reac-
tor (Hamilton, ON) and delivered as a [125I]Na in 0.1 mol/L NaOH
solution.
SPECT–CT imaging
Due to the rapid clearance of compound [123I]–7a and the high
tumour uptake of [125I]–7d, both 7a and 7d were then labelled
with 123I and used in a SPECT–CT imaging study. In both cases,
there was excellent tumour visualization and low nonspecific
binding (Fig. 5), which was consistent with the biodistribution
data at 24 h. In addition to the tumour, the eyes were also visible
due to the presence of melanin; an observation also in agreement
with the biodistribution data and literature reports for other
benzamides.15
Conclusions
A convenient synthetic method to prepare a new class of tria-
zole-based radioiodinated benzamide compounds capable of tar-
geting melanin-expressing tumors in vivo was developed. Compounds
were labelled in modest to high yields with 123I or 125I, and ob-
tained in >95% radiochemical purity prior to evaluation in vivo
using a murine melanoma tumour model. Two promising com-
pounds were identified, and SPECT–CT imaging studies showed
clear tumour uptake and clearance from nontarget organs. As the
lead compound, 7d represents a new melanin-binding radiophar-
maceutical that can be used for treating metastatic melanoma
alone or in combination with immunotherapies or as a diagnostic
agent to assess the impact of new treatments.
Synthesis
TAAG constructs
Compounds 1, 2, and 3 were synthesized according to literature
Experimental
procedures.19
General methods and materials
1H and 13C NMR spectra were recorded on a Bruker AV600 spec-
General procedure 1: preparation of protected amines (4e–4i)
To a solution of 4-(N-Boc-amino)piperidine (1 equiv) in DCM was
added the appropriate benzylhalide (1.2 equiv) and DIPEA (1.5 equiv),
and the reaction was stirred at room temperature for 24 h. The
reaction mixture was concentrated and purified through column
chromatography on silica with 1:1 hexane:ethyl acetate contain-
ing 1% triethylamine.
1
trometer. H and 13C NMR chemical shifts are expressed in parts
per million (ppm), and coupling constants are expressed in Hertz
(Hz). High resolution mass spectra (HRMS) were recorded on a
Waters Micromass Q-Tof Global mass spectrometer using electro-
spray ionization (ESI). Infrared (IR) spectra were obtained on a
Bio-Rad FTS-40 FT-IR spectrometer. High performance liquid chro-
matography (HPLC) was performed using a Varian ProStar model
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4e tert-Butyl 1-(3,5-dimethoxybenzyl)piperidin-4-ylcarbamate
Following general procedure 1, the product was isolated as a
white powder (51%, 0.160 g yield). 1H NMR (CDCl3, 600 MHz): ␦ 6.48
(d, 2H, J = 1.8 Hz), 6.35 (t, 1H, J = 2.2 Hz), 4.43 (s, 1H), 3.78 (s, 6H),
3.47(s, 1H) 3.41 (s, 2H), 2.78 (m, 2H), 2.08 (m, 2H), 1.90 (m, 2H), 1.44
(s, 11H). 13C NMR (CDCl3, 150 MHz): ␦ 160.7, 155.1, 141.1, 106.8, 98.9,
79.2, 63.1, 55.3, 52.4, 47.8, 32.6, 28.4. HRMS–ESI (m/z): [M + H]+ calcd
for C19H30N2O4H, 351.2284; found, 351.2278. mp 74–77 °C dec.
5g 1-(2-Fluorobenzyl)piperidin-4-amine
Following general procedure 2, the product was isolated as a
yellow oil (39%, 0.078 g yield). 1H NMR (CDCl3, 600 MHz): ␦ 7.35 (td,
1H, J = 1.6, 7.5 Hz), 7.21 (m, 1H), 7.08 (td, 1H, J = 0.9, 7.5 Hz), 7.00 (m,
1H), 3.56 (s, 2H), 2.84 (m, 2H), 2.64 (m, 1H), 2.07 (td, 2H, J = 2.0,
11.6 Hz), 1.78 (m, 2H), 1.71 (s, 2H), 1.39 (m, 2H). 13C NMR (CDCl3,
150 MHz): ␦ 161.4 (d, JCF = 245.9 Hz), 131.5 (d, JCF = 4.1 Hz), 128.6 (d,
J
J
CF = 8.2 Hz), 125.0 (d, JCF = 14.3 Hz), 123.8 (d, JCF = 2.6 Hz), 115.2 (d,
CF = 22.4 Hz), 55.2, 52.2, 48.6, 35.8.
4f tert-Butyl 1-(3,5-dinitrobenzyl)piperidin-4-ylcarbamate
Following general procedure 1, the product was isolated as a
yellow solid (92%, 0.279 g yield). 1H NMR (CDCl3, 600 MHz): ␦ 8.92
(s, 1H), 8.54 (s, 2H), 4.46 (s, 1H), 3.67 (s, 2H), 3.51 (m, 1H), 2.77 (m, 2H),
2.23 (m, 2H), 1.95 (m, 2H), 1.44 (s, 11H). 13C NMR (CDCl3, 150 MHz):
␦ 155.2, 148.6, 144.3, 128.6, 117.7, 79.4, 61.3, 52.5, 47.5, 32.5, 28.4.
HRMS–ESI (m/z): [M + H]+ calcd for C17H24N4O6H, 381.1774; found,
381.1770. mp 145–148 °C dec.
5h 1-(3-Fluorobenzyl)piperidin-4-amine
Following general procedure 2, the product was isolated as a
yellow oil (35%, 0.032 g yield). 1H NMR (CDCl3, 600 MHz): ␦ 7.24 (m,
1H), 7.05 (m, 2H), 6.91 (m, 1H), 3.46 (s, 2H), 2.79 (m, 2H), 2.66 (m, 1H),
2.01 (m, 2H), 1.78 (m, 2H), 1.59 (s, 2H), 1.38 (m, 2H). 13C NMR (CDCl3,
150 MHz): ␦ 162.9 (d, JCF = 245.6 Hz), 141.5 (d, JCF = 7.11 Hz) 129.5 (d,
J
CF = 8.32 Hz), 124.4, 115.6 (d, JCF = 21.27 Hz), 113.8 (d, JCF = 21.27 Hz),
62.4, 52.4, 48.7, 35.9.
4g tert-Butyl 1-(2-fluorobenzyl)piperidin-4-ylcarbamate
Following general procedure 1, the product was isolated as a
white solid (93%, 0.296 g yield). 1H NMR (CDCl3, 600 MHz): ␦ 7.38
(m, 1H), 7.24 (m, 1H), 7.11 (m, 1H), 7.02 (m, 1H), 4.42 (s, 1H), 3.59 (s,
2H), 3.47 (m, 1H), 2.84 (m, 2H), 2.19 (m, 2H), 1.92 (m, 2H), 1.43 (s,
11H). 13C NMR (CDCl3, 150 MHz): ␦ 161.4 (d, JCF = 247.6 Hz), 155.2,
131.7, 128.9, 123.5, 115.3 (d, JCF = 22.3 Hz), 79.3, 55.1, 52.0, 47.5, 32.4,
28.4.
5i 1-(4-Fluorobenzyl)piperidin-4-amine
Following general procedure 2, the product was isolated as a
yellow oil (41%, 0.055 g yield). 1H NMR (CDCl3, 600 MHz): ␦ 7.26 (m,
2H), 6.98 (m, 2H), 3.44 (s, 2H), 2.79 (m, 2H), 2.66 (m, 1H), 1.99 (m,
2H), 1.77 (m, 4H), 1.38 (m, 2H). 13C NMR (CDCl3, 150 MHz): ␦ 161.9 (d,
J
J
CF = 243.4 Hz), 134.3 (d, JCF = 2.4 Hz), 130.5 (d, JCF = 7.5 Hz), 114.9 (d,
CF = 21.1 Hz), 62.2, 52.3, 48.7, 35.8, 29.7.
Procedures to prepare 6a, 6b, and 6c from alternate TAAG constructs
4h tert-Butyl 1-(3-fluorobenzyl)piperidin-4-ylcarbamate
6a N-(2-(Diethylamino)ethyl)-2-(4-(4,4,5,5,6,6,7,7,8,8,9,9,9-
tridecafluorononyl)-1H-1,2,3-triazol-1-yl)acetamide
Following general procedure 1, the product was isolated as a
white solid (77%, 0.180 g yield). H NMR (CDCl3, 600 MHz): ␦ 7.25
1
To a solution of 1, the tin TAAG methyl ester (1 equiv) in meth-
anol (1 mL), was added N,N-diethylethylenediamine (3 equiv) and
DIPEA (3 equiv), and the reaction was heated to 60 °C for 2 h. The
reaction mixture was concentrated and purified through column
chromatography on silica with 20:1 dichloromethane:methanol.
The fractions containing the desired compound were concen-
trated, and the product was isolated as a clear oil (88%, 0.052 g
yield). 1H NMR (MeOD, 600 MHz): ␦ 8.08 (s, 1H), 5.24 (s, 2H), 3.44 (t,
2H, J = 6.8 Hz), 2.82 (m, 6H), 2.47 (m, 6H), 1.40 (t, 6H, J = 8.4, 30.2 Hz),
1.13 (m, 6H). 13C NMR (MeOD, 150 MHz): ␦ 168.2, 143.3, 134.2, 52.4,
52.0, 48.0, 37.0, 28.3 (t, JCSn = 22.9 Hz), 10.4, 0.0 (t, JCSn = 197.9 Hz).
(m, 1H), 7.05 (m, 2H), 6.92 (m, 1H), 4.47 (s, 1H), 3.46 (s, 3H), 2.77 (m,
2H), 2.09 (m, 2H), 1.90 (m, 2H), 1.44 (m, 11H). 13C NMR (CDCl3,
150 MHz): ␦ 163.1 (d, JCF = 245.0 Hz), 155.2, 141.4 (d, JCF = 5.4 Hz),
129.6 (d, JCF = 8.6 Hz), 124.4, 115.6 (d, JCF = 21.4 Hz), 113.9 (d, JCF
=
21.4 Hz), 79.2, 62.4, 52.4, 47.7, 32.6, 28.4.
4i tert-Butyl 1-(4-fluorobenzyl)piperidin-4-ylcarbamate
Following general procedure 1, the product was isolated as a
1
white solid (65%, 0.209 g yield). H NMR (CDCl3, 600 MHz): ␦ 7.27
(m, 2H), 6.99 (m, 2H), 4.43 (s, 1H), 3.48 (m, 1H), 3.45 (s, 2H), 2.78 (m,
2H), 2.09 (m, 2H), 1.91 (m, 2H), 1.14 (s, 11H). 13C NMR (CDCl3,
150 MHz): ␦ 162.0 (d, JCF = 243.5 Hz), 155.2, 130.6 (d, JCF = 7.0 Hz),
115.1 (d, JCF = 21.0 Hz), 79.3, 62.2, 52.2, 47.7, 32.5, 28.4.
IR (KBr, disc) ␷¯
(cm−1): 3324, 2973, 1675 cm−1. HRMS–ESI (m/z): [M +
H]+ calcd for C34H30N5OF39Sn, 1386.0935; found, 1386.0883. HPLC
Rt = 18.5 min.
General procedure 2: deprotection of protected amines (5e–5i)
To a solution of Boc protected amine (1 equiv) in DCM was added
TFA (10 equiv), and the reaction was stirred at room temperature
overnight. The reaction mixture was concentrated, dissolved in
10 mL of saturated sodium bicarbonate, and extracted three times
with 10 mL of DCM (Scheme 2, part 2). The organic layer was dried
over Na2SO4 and concentrated to give the desired free amine.
6b N-(2-Diethylamino-ethyl)-2-{5-phenyl–4-[tris-(3,3,4,4,5,5,6,6,
7,7,8,8,8-tridecafluoro-octyl)-stannanyl]-[1,2,3]triazol–1-yl}-acetamide
To a solution of 2, the phenyl-tin TAAG methyl ester (1 equiv) in
methanol (1 mL), was added N,N-diethylethylenediamine (3 equiv)
and DIPEA (3 equiv), and the reaction was heated to 60 °C for
3 days. The reaction mixture was concentrated and purified
throughcolumnchromatographyonsilicawith20:1dichlorometh-
ane:methanol. The fractions containing the desired compound
were concentrated, and the product was isolated as a clear oil
(19%, 0.012 g yield). 1H NMR (CDCl3, 600 MHz): ␦ 7.49 (m, 3H), 7.28
(m, 2H), 4.92 (s, 2H), 3.36 (q, 2H, J = 5.6, 11.1 Hz), 2.60 (m, 6H), 2.18
(m, 6H), 1.18 (m, 6H), 1.01 (t, 6H, J = 7.1 Hz). 13C NMR (CDCl3,
150 MHz): ␦ 166.2, 146.8, 142.8, 131.2, 130.1, 129.9, 127.9, 51.9, 51.5,
47.5, 37.3, 28.1 (t, JCSn = 23.0 Hz), 11.8, 0.0 (t, JCSn = 188.0 Hz). IR (KBr,
5e 1-(3,5-Dimethoxybenzyl)piperidin-4-amine
Following general procedure 2, the product was isolated as a
yellow oil (93%, 0.072 g yield). 1H NMR (CDCl3, 600 MHz): ␦ 6.48 (s,
2H), 6.34 (s, 1H), 3.77 (s, 6H), 3.42 (s, 2H), 2.82 (m, 2H), 2.64 (m, 1H),
2.01 (m, 2H), 1.77 (m, 2H), 1.47 (s, 2H), 1.38 (m, 2H). 13C NMR (CDCl3,
150 MHz): ␦ 160.7, 141.2, 106.8, 98.9, 63.1, 55.3, 52.5, 48.8, 36.0.
disc) ␷¯
(cm−1): 3286, 2929, 1683. HRMS–ESI (m/z): [M + H]+ calcd for
C40H34N5OF39SnH, 1462.1250; found, 1462.1219. HPLC Rt = 19.10 min.
5f 1-(3,5-Dinitrobenzyl)piperidin-4-amine
Following general procedure 2, the product was isolated as an
orange oil (89%, 0.178 g yield). 1H NMR (CDCl3, 600 MHz): ␦ 8.93 (t,
1H, J = 2.05 Hz), 8.54 (d, 2H, J = 1.98 Hz), 3.68 (s, 2H), 2.80 (m, 3H),
2.18 (td, 2H, J = 2.12, 11.76 Hz), 1.87 (m, 6H), 1.50 (m, 2H). 13C NMR
(CDCl3, 150 MHz): ␦ 148.6, 144.2, 128.6, 117.6, 61.3, 52.3, 48.4, 35.0.
HRMS–ESI (m/z): [M + H]+ calcd for C12H16N4O4H, 281.1250; found,
281.1262.
6c N-(2-(Piperidin-1-yl)ethyl)-2-(4-(tributylstannyl)-1H-1,2,3-triazol-
1-yl)acetamide
To a solution of 3, the tin TAAG methyl ester (1 equiv) in meth-
anol (1 mL), was added 1-(2-aminoethyl)piperidine (3 equiv) and
DIPEA (3 equiv), and the reaction was heated to 60 °C for 3 days.
The reaction mixture was concentrated and purified through column
Published by NRC Research Press

Rathmann et al.
779
chromatography on silica with 20:1 dichloromethane:methanol.
The fractions containing the desired compound were concen-
trated, and the product was isolated as a yellow solid (76%, 0.089 g
yield). 1H NMR (CDCl3, 600 MHz): ␦ 7.61 (s, 1H), 7.11 (s, 1H), 5.11 (s,
2H), 3.40 (q, 2H, J = 5.9, 11.8 Hz), 2.56 (t, 2H, J = 5.9 Hz), 2.50 (s, 4H),
1.60 (m, 4H), 1.53 (m, 6H), 1.46 (s, 2H), 1.33 (m, 6H), 1.11 (m, 6H, J =
8.23, 26.9 Hz), 0.88 (t, 9H, J = 7.4 Hz); 13C NMR (CDCl3, 150 MHz): ␦
165.9, 145.2, 131.4 (t, JC,Sn = 37.0 Hz), 56.4, 54.1, 52.4, 35.7, 29.0 (t,
9H, J = 7.4 Hz). 13C NMR (CDCl3, 150 MHz): ␦ 164.9, 161.3 (d, JCF
=
246.5 Hz), 145.7, 131.4 (t, JCSn = 36.9 Hz), 128.8 (d, JCF = 7.6 Hz), 123.8,
115.2 (d, JCF = 22.2 Hz), 55.1, 52.7, 51.7, 46.8, 31.7, 29.0 (t, JCSn = 10.4),
27.2 (t, JCSn = 29.2 Hz), 13.6, 10.0 (t, JCSn = 178.3 Hz). IR (KBr, disc) ␷
¯
(cm–1): 3279, 3094, 2926, 2871, 2852, 2798, 1653, 1557, 1489, 1456, 1431,
1367, 1341, 1321, 1287. HRMS–ESI (m/z): [M + H]+ calcd for C28H46N5OSnFH,
608.2792; found, 608.2783. Rf (10:1 dichloromethane:methanol) = 0.56.
6h N-(1-(3-Fluorobenzyl)piperidin-4-yl)-2-(4-(tributylstannyl)-
1H-1,2,3-triazol-1-yl)acetamide
J
C,Sn = 10.2 Hz), 27.2 (t, JC,Sn = 29.9 Hz), 25.1, 23.7, 13.6, 9.9 (t, JC,Sn =
176.4 Hz). IR (KBr, disc) ␷¯
(cm–1): 3301, 2931, 2871, 2853, 1684, 1559,
1540, 1457. HRMS–ESI (m/z): [M + H]+ calcd for C23H46N5OSn, 528.2729;
Following general procedure 3, the product was isolated as a
yellow solid (20%, 0.014 g yield). 1H NMR (CDCl3, 600 MHz): ␦ 7.58 (s,
1H), 7.23 (m, 1H), 7.02 (m, 2H), 6.91 (td, 1H, J = 2.1, 8.7 Hz), 6.05 (s,
1H), 5.06 (s, 2H), 3.78 (m, 1H), 3.44 (s, 2H), 2.69 (m, 2H), 2.10 (t, 2H,
J = 10.6 Hz), 1.82 (m, 2H), 1.54 (m, 6H), 1.40 (m, 2H), 1.32 (m, 6H), 1.13
(m, 6H), 0.88 (t, 9H, J = 7.4 Hz). 13C NMR (CDCl3, 150 MHz): ␦ 164.9,
found, 528.2735.
General procedure 3: coupling amines to TAAG (6d–6i)
To a solution of tin TAAG methyl ester (3) (1 equiv) in methanol
(1 mL) was added amine (1.2 equiv) and DIPEA (3 equiv), and the
reaction was heated to 60 °C for 3 days. The reaction mixture was
concentrated and purified through column chromatography on
silica with 20:1 dichloromethane:methanol. The fractions con-
taining the desired compound were concentrated to give the de-
sired radiolabelling precursor.
163.0 (JCF = 246.1 Hz), 145.7, 141.2, 131.4 (JCF = 36.2 Hz), 129.6 (JCF
=
8.0 Hz), 124.3, 115.5 (JCF = 21.4 Hz), 113.9 (JCF = 20.9 Hz), 62.3, 52.7,
51.9, 46.9, 31.7, 29.0 (JCSn = 10.7 Hz), 27.2 (JCSn = 29.6 Hz), 13.6, 10.0
(JCSn = 176.8 Hz). IR (KBr, disc) ␷¯
(cm–1): 3279, 3092, 2955, 2927, 2871,
2852, 1653, 1556, 1485, 1454, 1293, 1255. HRMS–ESI (m/z): [M + H]+
calcd for C28H46N5OSnFH, 608.2792; found, 608.2781. Rf (10:1
dichloromethane:methanol) = 0.48.
6d N-(1-Benzylpiperidin-4-yl)-2-(4-(tributylstannyl)-1H-1,2,3-triazol-
1-yl)acetamide
Following general procedure 3, the product was isolated as a
white solid (64%, 0.165 g yield). 1H NMR (CDCl3, 600 MHz): ␦ 7.57 (s,
1H), 7.29 (m, 4H), 7.23 (m, 1H), 6.08 (bs, 1H), 5.07 (s, 2H), 3.80 (m, 1H),
3.49 (s, 2H), 2.75 (m, 2H), 2.13 (m, 2H) 1.83 (m, 2H), 1.55 (m, 6H), 1.43
(m, 2H), 1.34 (m, 6H), 1.15 (m, 6H), 0.89 (t, 9H, J = 7.4 Hz). 13C NMR
(CDCl3, 150 MHz): ␦ 164.9, 145.7, 131.4, 129.1, 128.3, 127.2, 62.8, 52.7,
51.8, 46.8, 31.6, 29.0 (t, JCSn = 10.9 Hz), 27.2 (t, JCSn = 29.8 Hz), 13.7,
6i N-(1-(4-Fluorobenzyl)piperidin-4-yl)-2-(4-(tributylstannyl)-
1H-1,2,3-triazol-1-yl)acetamide
Following general procedure 3, the product was isolated as a
white solid (18%, 0.036 g yield). 1H NMR (CDCl3, 600 MHz): ␦ 7.58 (s,
1H), 7.26 (s, 2H), 6.99 (t, 2H, J = 8.8 Hz), 6.26 (s, 1H), 5.08 (s, 2H), 3.81
(m, 1H), 3.51 (s, 2H), 2.79 (m, 2H), 2.16 (s, 2H), 1.85 (m, 2H), 1.54 (m,
6H), 1.49 (m, 2H), 1.32 (m, 6H), 1.13 (m, 6H), 0.87 (t, 9H, J = 7.1 Hz).
13C NMR (CDCl3, 150 MHz): ␦ 165.0, 162.1 (JCF = 21.3 Hz), 145.6, 133.1,
131.6 (JCSn = 35.4 Hz), 130.7, 115.1 (JCF = 21.3 Hz), 61.9, 52.6, 51.7, 46.7,
10.01 (t, JCSn = 177.1 Hz). IR (KBr, disc) ␷¯
(cm–1): 3297, 3092, 2954,
2927, 2871, 2851, 2804, 2754, 1653, 1558. HRMS–ESI (m/z): [M + H]+
calcd for C28H47N5OSnH, 590.2886; found, 590.2877.
31.3, 29.0 (JCSn = 10.5 Hz), 27.2 (JCSn = 29.3 Hz), 13.6, 10.0 (JCSn
=
176.3 Hz). IR (KBr, disc) ␷¯
(cm–1): 3292, 2955, 2924, 2852, 1669, 1603,
6e N-(1-(3,5-Dimethoxybenzyl)piperidin-4-yl)-2-(4-(tributylstannyl)-
1H-1,2,3-triazol-1-yl)acetamide
1551, 1510, 1464. HRMS–ESI (m/z): [M + H]+ calcd for C28H46N5OSnFH,
608.2792; found, 608.2794. Rf (10:1 dichloromethane:methanol) = 0.46.
Following general procedure 3, the product was isolated as an
orange solid (7%, 0.014 g yield). 1H NMR (CDCl3, 600 MHz): ␦ 7.58 (s,
1H), 6.48 (m, 2H), 6.35 (t, 1H, J = 2.2 Hz), 6.21 (s, 1H), 5.07 (s, 2H), 3.77
(s, 7H), 3.44 (s, 2H), 2.77 (m, 2H), 2.15 (m, 2H), 1.84 (m, 2H), 1.54 (m,
6H), 1.48 (m, 2H), 1.32 (m, 6H), 1.12 (m, 6H), 0.87 (t, 9H, J = 7.3 Hz).
13C NMR (CDCl3, 150 MHz): ␦ 164.8, 160.8, 145.6, 131.5, 99.3, 62.8,
Radiolabelling
General radioiodination procedure 1: labelling of 6a–6c
To a solution of a tin precursor (100 ␮g, 0.068–0.190 ␮mol) in
methanol (100 ␮L) was added iodogen (25 ␮g) and acetic acid (5 ␮L),
in the presence of no carrier added [123I/125I]-iodine (2.9 MBq –
196.5 MBq). The reaction was placed on the shaker for 10 min, at
which point it was quenched with Na2S2O3 (0.1 mol/L, 100 ␮L). The
desired product was isolated by HPLC, concentrated to dryness,
and formulated in PBS for biological studies.
55.3, 52.7, 51.9, 46.8, 31.4, 29.0 (t, JCSn = 10.7 Hz), 27.2 (t, JCSn
=
29.1 Hz), 13.6, 10.0 (t, JCSn = 176.8 Hz). IR (KBr, disc) ␷
¯
(cm–1): 3291,
2955, 2954, 2851, 1669, 1597, 1552, 1463, 1430, 1366, 1341. HRMS–
ESI (m/z): [M + H]+ calcd for C30H51N5O3SnH, 650.3098; found,
650.3111. mp 101–104 °C dec.
6f N-(1-(3,5-Dinitrobenzyl)piperidin-4-yl)-2-(4-(tributylstannyl)-
1H-1,2,3-triazol-1-yl)acetamide
General radioiodination procedure 2: labelling of 6d–6i
To a solution of a tin precursor (100 ␮g, 0.147–0.170 ␮mol) in
chloroform (100 ␮L) was added iodogen (25 ␮g) in chloroform
(25 ␮L) and acetic acid (5 ␮L), in the presence of no carrier added
Following general procedure 3, the product was isolated as an
orange solid (9%, 0.036 g yield). 1H NMR (CDCl3, 600 MHz): ␦ 8.92 (s,
1H), 8.51 (s, 2H), 7.57 (s, 1H), 6.09 (m, 1H), 5.06 (s, 2H), 3.82 (m, 1H),
3.65 (s, 2H), 2.70 (m, 2H), 2.23 (t, 2H, J = 10.9 Hz), 1.87 (m, 2H), 1.55
(m, 6H), 1.45 (m, 2H), 1.32 (m, 6H), 1.14 (m, 6H), 0.88 (t, 9H, J =
7.34 Hz). 13C NMR (CDCl3, 150 MHz): ␦ 165.0, 148.6, 145.8, 144.0,
131.5, 128.5, 117.7, 61.3, 52.7, 52.1, 46.6, 31.6, 29.0 (t, JCSn = 10.3 Hz),
[
123I/125I]-iodine (4.4 MBq – 81.4 MBq). The reaction solution was
placed on the shaker for 10 min, at which point it was quenched
with Na2S2O5 (0.1 mol/L, 100 ␮L). The aqueous layer of the reaction
mixture was separate, and the desired product was isolated by
HPLC, concentrated to dryness, and formulated in PBS for biolog-
ical studies.
27.2 (t, JCSn = 29.4 Hz), 13.7, 10.0 (t, JCSn = 176.7 Hz). IR (KBr, disc) ␷
¯
(cm–1): 3263, 2954, 2925, 2852, 1656, 1539, 1459, 1344. HRMS–ESI
(m/z): [M + H]+ calcd for C28H45N5O3SnFH, 680.2588; found, 680.2592.
Lipophilicity measurements
The lipophilicity (log P) values were determined by the shake
flask method at pH 7.4.25 The results are summarized in Table 1.
6g N-(1-(2-Fluorobenzyl)piperidin-4-yl)-2-(4-(tributylstannyl)-
1H-1,2,3-triazol-1-yl)acetamide
Following general procedure 3, the product was isolated as an
orange oil (32%, 0.056 g yield). 1H NMR (CDCl3, 600 MHz): ␦ 7.33 (m,
1H), 7.21 (m, 1H), 7.07 (m, 1H), 7.00 (m, 1H), 6.04 (s, 1H), 5.05 (s, 2H),
3.76 (m, 1H), 3.53 (s, 2H), 2.74 (m, 2H), 2.15 (t, 2H, J = 10.9 Hz), 1.82
(m, 2H), 1.54 (m, 6H), 1.39 (m, 2H), 1.32 (m, 6H), 1.12 (m, 6H), 0.87 (t,
Mouse melanoma tumour model
B16F1 cells derived from mouse melanoma were purchased from
ATCC (CRL-6323). Cells were propagated using DMEM media, sup-
plemented with 10% fetal bovine serum (FBS) and 1% penicillin-
streptomycin, and grown at 37 °C with 5% CO2. Cells were used
Published by NRC Research Press

780
Can. J. Chem. Vol. 00, 0000
between passage numbers 7–28. Female C57Bl/6 mice were pur-
chased from Charles River (Senneville, QC) and housed under SPF
conditions in an established animal facility, with 12 h light and 12 h
dark cycles and with food and water ad libitum. Mice were injected
with 4.5 × 105 B16F1 cells in DPBS subcutaneously into the flank to
generate syngeneic melanoma tumours. Animal studies were ap-
proved by the Animal Research Ethics Board at McMaster University
in accordance with Canadian Council on Animal Care guidelines.
References
(1) National Cancer Institute. SEER Stat Fact Sheets: Melanoma of the Skin
[Online]. 2015. http://seer.cancer.gov/statfacts/html/melan.html.
(2) Johnson, D. B.; Sosman, J. A. Curr. Treat. Options Oncol. 2013, 14, 280. doi:10.
1007/s11864-013-0226-8.
(3) Chapman, P. B.; Hauschild, A.; Robert, C.; Haanen, J. B.; Ascierto, P.;
Larkin, J.; Dummer, R.; Garbe, C.; Testori, A.; Maio, M.; Hogg, D.; Lorigan, P.;
Lebbe, C.; Jouary, T.; Schadendorf, D.; Ribas, A.; O’Day, S. J.; Sosman, J. A.;
Kirkwood, J. M.; Eggermont, A. M.; Dreno, B.; Nolop, K.; Li, J.; Nelson, B.;
Hou, J.; Lee, R. J.; Flaherty, K. T.; McArthur, G. A. N. Engl. J. Med. 2011, 364,
2507. doi:10.1056/NEJMoa1103782.
(4) Lidsky, M.; Antoun, G.; Speicher, P.; Adams, B.; Turley, R.; Augustine, C.; Tyler, D.;
Ali-Osman, F. J. Biol. Chem. 2014, 289, 27714. doi:10.1074/jbc.M113.532432.
(5) Villanueva, J.; Vultur, A.; Lee, J. T.; Somasundaram, R.; Fukunaga-Kalabis, M.;
Cipolla, A. K.; Wubbenhorst, B.; Xu, X.; Gimotty, P. A.; Kee, D.;
Santiago-Walker, A. E.; Letrero, R.; D’Andrea, K.; Pushparajan, A.; Hayden, J. E.;
Brown, K. D.; Laquerre, S.; McArthur, G. A.; Sosman, J. A.; Nathanson, K. L.;
Herlyn, M. Cancer Cell 2010, 18, 683. doi:10.1016/j.ccr.2010.11.023.
(6) Lebbe, C.; Weber, J. S.; Maio, M.; Neyns, B.; Harmankaya, K.; Hamid, O.;
O’Day, S. J.; Konto, C.; Cykowski, L.; McHenry, M. B.; Wolchok, J. D. Ann.
Oncol. 2014, 25, 2277. doi:10.1093/annonc/mdu441.
(7) Topalian, S. L.; Hodi, F. S.; Brahmer, J. R.; Gettinger, S. N.; Smith, D. C.;
McDermott, D. F.; Powderly, J. D.; Carvajal, R. D.; Sosman, J. A.; Atkins, M. B.;
Leming, P. D.; Spigel, D. R.; Antonia, S. J.; Horn, L.; Drake, C. G.;
Pardoll, D. M.; Chen, L.; Sharfman, W. H.; Anders, R. A.; Taube, J. M.;
McMiller, T. L.; Xu, H.; Korman, A. J.; Jure-Kunkel, M.; Agrawal, S.;
McDonald, D.; Kollia, G. D.; Gupta, A.; Wigginton, J. M.; Sznol, M. N. Engl. J.
Med. 2012, 366, 2443. doi:10.1056/NEJMoa1200690.
Biodistribution studies
Biodistribution studies were performed on C57Bl/6 mice bear-
ing B16F1 tumours at 10 days post inoculation (n = 5 for [123I]–7a, n = 4
for [125I]–7b, and n = 3 for the remaining seven compounds). Mice
were injected with 0.48 MBq [123I]–7a, 0.098 MBq [125I]–7b, 0.15 MBq
[
125I]–7c, 0.22 MBq [125I]–7d, 0.11 MBq [125I]–7e, 0.22 MBq [125I]–7f,
0.25 MBq [125I]–7g, 0.24 MBq [125I]–7h, or 0.25 MBq [125I]–7i. Twenty-
four hours after injection, animals were anesthetized with 3%
isoflurane and euthanized by cervical dislocation. Blood, adipose,
adrenals, bone, brain, eyes, gall bladder, heart, kidneys, large
intestine and caecum (with contents), liver, lungs, skeletal mus-
cle, small intestine (with contents), spleen, stomach (with con-
tents), thyroid and (or) trachea, B16F1 tumour, urine + bladder,
and tail were collected, weighed, and counted in a PerkinElmer
Wizard 1470 Automatic Gamma Counter. Decay correction was
used to normalize organ activity measurements to the time of
dose preparation for final data calculations of percent injected
dose per gram of tissue and (or) fluid (%ID/g).
(8) Wolchok, J. D.; Kluger, H.; Callahan, M. K.; Postow, M. A.; Rizvi, N. A.;
Lesokhin, A. M.; Segal, N. H.; Ariyan, C. E.; Gordon, R.-A.; Reed, K.;
Burke, M. M.; Caldwell, A.; Kronenberg, S. A.; Agunwamba, B. U.; Zhang, X.;
Lowy, I.; Inzunza, H. D.; Feely, W.; Horak, C. E.; Hong, Q.; Korman, A. J.;
Wigginton, J. M.; Gupta, A.; Sznol, M. N. Engl. J. Med. 2013, 369, 122. doi:10.
1056/NEJMoa1302369.
(9) Koch, S. E.; Lange, J. R. J. Am. Acad. Dermatol. 2000, 42, 731. doi:10.1067/mjd.
2000.103981.
SPECT–CT imaging
Imaging of [123I]–7a and [123I]–7d was completed using female
C57Bl/6 mice bearing B16F1 tumours, 10 days after inoculation.
The mice were administered 200 ␮L of PBS containing [123I]–7a
(ϳ24.1 MBq) or [123I]–7d (ϳ24.9 MBq) via tail vein injection. For
(10) Joyal, J. L.; Barrett, J. A.; Marquis, J. C.; Chen, J.; Hillier, S. M.; Maresca, K. P.;
Boyd, M.; Gage, K.; Nimmagadda, S.; Kronauge, J. F.; Friebe, M.;
Dinkelborg, L.; Stubbs, J. B.; Stabin, M. G.; Mairs, R.; Pomper, M. G.;
Babich, J. W. Cancer Res. 2010, 70, 4045. doi:10.1158/0008-5472.CAN-09-4414.
(11) Michelot, J. M.; Moreau, M. F.; Veyre, A. J.; Bonafous, J. F.; Bacin, F. J.;
Madelmont, J. C.; Bussiere, F.; Souteyrand, P. A.; Mauclaire, L. P.;
Chossat, F. M.; Papon, J. M.; Labarre, P.G.; Kauffmann, P.; Plagne, R.J. J. Nucl.
Med. 1993, 34, 1260. PMID:8326382.
(12) Bonnet-Duquennoy, M.; Papon, J.; Mishellany, F.; Labarre, P.; Guerquin-Kern, J.-L.;
Wu, T.-D.; Gardette, M.; Maublant, J.; Penault-Llorca, F.; Miot-Noirault, E.; Cayre, A.;
Madelmont, J.-C.; Chezal, J.-M.; Moins, N. Int. J. Cancer 2009, 125, 708. doi:10.1002/ijc.
24413.
(13) Labarre, P.; Papon, J.; Moreau, M.-F.; Moins, N.; Veyre, A.; Madelmont, J.-C. Eur. J.
Nucl. Med. Mol. Imaging 1999, 26, 494. doi:10.1007/s002590050416.
(14) Silberstein, E. B. Semin. Nucl. Med. 2012, 42, 164. doi:10.1053/j.semnuclmed.
2011.12.002.
(15) Michelot, J. M.; Moreau, M. F.; Labarre, P. G.; Madelmont, J. C.; Veyre, A. J.;
Papon, J. M.; Parry, D. F.; Bonafous, J. F.; Boire, J. Y.; Desplanches, G. G.;
Bertrand, S.J.; Meyniel, G. J. Nucl. Med. 1991, 32, 1573. PMID:1869982.
(16) Mier, W.; Kratochwil, C.; Hassel, J. C.; Giesel, F. L.; Beijer, B.; Babich, J. W.;
Friebe, M.; Eisenhut, M.; Enk, A.; Haberkorn, U. J. Nucl. Med. 2014, 55, 9.
doi:10.2967/jnumed.112.112789.
(17) Chang, C.-C.; Chang, C.-H.; Shen, C.-C.; Chen, C.-L.; Liu, R.-S.; Lin, M.-H.;
Wang, H.-E. Bioorg. Med. Chem. 2015, 23, 2261. doi:10.1016/j.bmc.2015.02.017.
(18) Liu, H.; Liu, S.; Miao, Z.; Deng, Z.; Shen, B.; Hong, X.; Cheng, Z. J. Med. Chem.
2013, 56, 895. doi:10.1021/jm301740k.
[
123I]–7a, the total amount of activity in the animal was low, so the
animal was euthanized prior to imaging to allow for longer acqui-
sition times. Prior to imaging for [123I]–7d, the mouse was anaes-
thetized with 2.5% isoflurane and maintained under the same
conditions for the length of the SPECT and CT scans. SPECT acqui-
sitions were completed for 32 frames (300 sec/frame for [123I]–7a
and 180 sec/frame for [123I]–7d) on a Gamma Medica Ideas X-SPECT
system (North Ridge, CA). CT acquisition consisted of 512 projec-
tions acquired over 360° with 75Kvp, 205 mA cone beam CT sys-
tem. Cobra Exxim software (Feldkamp filtered back-projection
cone beam reconstruction software) was used to reconstruct the
images at a voxel size of 155 ␮m and a matrix size of 5123. An
OS-EM interactive reconstructed method (two iterations per eight
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 the images.
(19) Darwish, A.; Blacker, M.; Janzen, N.; Rathmann, S. M.; Czorny, S.;
Hillier, S. M.; Joyal, J. L.; Babich, J. W.; Valliant, J. F. ACS Med. Chem. Lett. 2012,
3, 313. doi:10.1021/ml300003v.
(20) Gladysz, J. A.; Curran, D. P.; Horváth, I. T. Handbook of Fluorous Chemistry;
Wiley-VCH: Weinheim, 2004. doi:10.1002/3527603905.
(21) Zea-Ponce, Y.; Baldwin, R. M.; Zoghbi, S. S.; Innis, R. B. Appl. Radiat. Isot. 1994,
45, 63. doi:10.1016/0969-8043(94)90149-X.
Supplementary material
Supplementary data are available with the article through the
journal Web site at http://nrcresearchpress.com/doi/suppl/10.1139/
cjc-2016-0239.
(22) Ings, R. M. J. Drug Metab. Rev. 1984, 15, 1183. doi:10.3109/03602538409033561.
PMID:6396056.
(23) Moins, N.; Papon, J.; Seguin, H.; Gardette, D.; Moreau, M.-F.; Labarre, P.;
Bayle, M.; Michelot, J.; Gramain, J.-C.; Madelmont, J.-C.; Veyre, A. Nucl. Med.
Biol. 2001, 28, 799. doi:10.1016/S0969-8051(01)00242-6.
(24) Böhm, H.-J.; Banner, D.; Bendels, S.; Kansy, M.; Kuhn, B.; Müller, K.;
Obst-Sander, U.; Stahl, M. Chembiochem 2004, 5, 637. doi:10.1002/cbic.
200301023.
(25) Wilson, A. A.; Jin, L.; Garcia, A.; DaSilva, J. N.; Houle, S. Appl. Radiat. Isot.
2001, 54, 203. doi:10.1016/S0969-8043(00)00269-4.
Acknowledgements
This work was supported by grants from the Natural Sciences
and Engineering Council of Canada and the Ontario Institute for
Cancer Research. X-ray crystallography support was provided by
Dr. Hilary Jenkins, McMaster University, who acquired data and
solved the crystal structure. The authors acknowledge the contri-
butions of Dr. Denis Snider towards the preparation and editing of
this manuscript.
Published by NRC Research Press