Synthesis and in Vivo Evaluation of [123I]Melanin-Targeted Agents

Article abstract of DOI:10.1021/acs.jmedchem.5b00777

This study reports the synthesis, [¹²³I]radiolabeling, and biological profile of a new series of iodinated compounds for potential translation to the corresponding [¹³¹I]radiolabeled compounds for radionuclide therapy of melanoma. Ra

Full text of DOI:10.1021/acs.jmedchem.5b00777

Article
pubs.acs.org/jmc
Synthesis and in Vivo Evaluation of [123I]Melanin-Targeted Agents
Maxine P. Roberts,†,§ Vu Nguyen,†,§ Mark E. Ashford,† Paula Berghofer,† Naomi A. Wyatt,†
Anwen M. Krause-Heuer,†,∥ Tien Q. Pham,† Stephen R. Taylor,†,⊥ Leena Hogan,† Cathy D. Jiang,†
Benjamin H. Fraser,† Nigel A. Lengkeek,† Lidia Matesic,† Marie-Claude Gregoire,† Delphine Denoyer,‡,#
Rodney J. Hicks,‡ Andrew Katsifis,†,∇ and Ivan Greguric*,†
†LifeSciences Division, Australian Nuclear Science and Technology Organisation (ANSTO), Locked Bag 2001, Kirrawee DC, New
South Wales 2232, Australia
‡Centre for Molecular Imaging, Peter MacCallum Cancer Centre, 12 St. Andrew’s Place, East Melbourne, Victoria 3002, Australia
S
* Supporting Information
ABSTRACT: This study reports the synthesis, [123I]radiolabeling, and biological profile of
a new series of iodinated compounds for potential translation to the corresponding
[
131I]radiolabeled compounds for radionuclide therapy of melanoma. Radiolabeling was
achieved via standard electrophilic iododestannylation in 60−90% radiochemical yield.
Preliminary SPECT imaging demonstrated high and distinct tumor uptake of all
compounds, as well as high tumor-to-background ratios compared to the literature
compound [123I]4 (ICF01012). The most favorable compounds ([123I]20, [123I]23, [123I]
41, and [123I]53) were selected for further biological investigation. Biodistribution studies
indicated that all four compounds bound to melanin containing tissue with low in vivo
deiodination; [123I]20 and [123I]53 in particular displayed high and prolonged tumor
uptake (13% ID/g at 48 h). [123I]53 had the most favorable overall profile of the
cumulative uptake over time of radiosensitive organs. Metabolite analysis of the four radiotracers found [123I]41 and [123I]53 to
be the most favorable, displaying high and prolonged amounts of intact tracer in melanin containing tissues, suggesting melanin
specific binding. Results herein suggest that compound [123I]53 displays favorable in vivo pharmacokinetics and stability and
hence is an ideal candidate to proceed with further preclinical [131I] therapeutic evaluation.
evaluation, followed by targeted radiopharmaceuticals for
treatment of malignant melanoma.
INTRODUCTION
■
Melanoma is an aggressive form of skin cancer with metastatic
tendencies and is currently the fourth most commonly
diagnosed cancer in Australia (excluding basal and squamous
cell carcinoma of the skin), following breast cancer in women
or prostate cancer in men and bowel and lung cancer.1,2
Despite the increasing incidence of this disease, surgical
excision with margins based on Breslow thickness and
anatomical location still remains the most effective treatment
of melanoma grades 1−3. Treatment by surgical resection is
limited once the melanoma has metastasized to secondary
locations (grade 4).3 Grade 4 melanotic tumors that exhibit
excessive melanin pigmentation presents an attractive target for
both diagnostic and therapeutic applications.4−6
A number of methods have been investigated for selective
delivery of various diagnostic and therapeutic radionuclides to
malignant melanoma; of these, numerous [123I] labeled
benzamide derivatives have demonstrated high uptake in
melanotic tissue.7−12 Studies of the first [123I] labeled
benzamides [123I]N-(2-diethylaminoethyl)-4-iodobenzamide
([123I]BZA, 1)11,12 and [123I]N-(2-diethylaminoethyl)-2-iodo-
benzamide ([123I]BZA2, 2)13,14 (Figure 1) found cell uptake to
be dependent on melanin content.15 Subsequent single-photon
emission computed tomography (SPECT) evaluation in
patients demonstrated a high affinity and selectivity in imaging
cutaneous and ocular melanoma deposits.11,14,16,17 The
substitution of the benzamide moiety in 1 and 2 for a
nicotinamide moiety was performed in [123I]MEL008 (3) to
enhance hydrophilicity and rapid clearance via renal ex-
cretion.18
Of the iodine radioisotopes, iodine-123 (123I) has the most
favorable properties for SPECT imaging purposes with a short
half-life (13.2 h), low patient radiation exposure and provides
the possibility of complementary radiolabeling of the same
compound with other isotopes, such as iodine-131 (131I).7,8
Iodine-131 is not an ideal isotope for imaging but is the most
commonly used radionuclide for therapy due to its relatively
long half-life (8.0 days) and emission of β-particles that
penetrate a few millimeters in tissue, making it useful for
treatment of solid tumors.6,9,10 Hence, [123I] and [131I] can be
used as complementary isotopes for initial broad biological
Other molecules reported to show in vivo selectivity for
melanin are those with various (hetero)aromatic systems.10,19
These melanin binding molecules that are taken up by the
tumor cells are not receptor mediated and do not directly
participate in the melanin biosynthesis pathway.18 As a
Received: May 21, 2015
© XXXX American Chemical Society
A
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main subgroups based on the structures of 1−5: the
benzylpiperazine group and the isoindolin-1-one heterocyclic
group (Figure 2).
Figure 1. Chemical structures of various radiotracers with melanin
specificity.
Figure 2. General structure of compounds containing (a) the
iodinated benzylpiperazine group and (b) the isoindolin-1-one
heterocyclic group of compounds synthesized in this study.
consequence, they do not appear to be transported into the cell
through any known active transporter.10,18,20,21 Although not
completely understood, a factor that may be responsible for
these types of compounds having high uptake into melanin
expressing tumors could include a component of radiotracer
bound to melanin and hence not released from the cell.
These types of analogues were found to bind to melanin
expressing tumors with high specificity and moderate-to-high
affinity in mice; particularly ICF01012 ([125I]4, Figure 1)
displayed highly specific and long-lasting uptake in the tumor.10
Furthermore, [131I], [123I] labeled,22 and multimodal [18F]/
For the first group of new compounds synthesized (Figure
2a), the benzylpiperazine moiety (of 5) was maintained because
of its known stability and ease of radioiodination.27 The
carboxylic acids for forming the acetamide were selected based
on structures identified in the previously discussed compounds
(Figure 1).10−14,18,25 This included the nicotinamide group
found in 3, 1,8-napthyridin-2-yl based compounds, which are
structurally similar to the quinoxalin-2-yl group found in 4, and
the isoindolin-1-one group similar to 5. Synthesis of these
compounds was achieved using standard literature coupling
conditions (HOBt, EDC, DIPEA) that were used previously for
the synthesis of similar compounds.18,25,27 Commercially
available 4-iodobenzylpiperazine (6) (for synthesis of iodoi-
nated nonradioactive standards) or 4-bromobenzylpiperazine
(7) (for synthesis of brominated compounds) was condensed
with nicotinic acid (8) to yield 9−10 and separately with 2-(1-
oxoisoindolin-2-yl)acetic acid (12) to yield 13 and 14 (Scheme
1, top). 13 and 14 (which contain both benzylpiperazine and
the isoindolin-1-one heterocyclic group) belong to both
synthetic subgroups but have been categorized with the
compounds of the benzylpiperazine group (Figure 2a) because
of the similar synthetic method.
[
131I] labeled23 analogues of 4 have shown suppression in
B16F023 and SK-MEL-322,24 tumor bearing mice. This further
validated the potential of these types of compounds for
therapeutic applications of metastatic melanoma and hence led
our research group to the development of [123I]MEL037 (5,
Figure 1), which demonstrated high and prolonged tumor
uptake, rapid whole body clearance, and potential therapeutic
application.25
The structural modifications undertaken for 5 included a
benzyl substituent (for straightforward radiolabeling via the
stannyl precursor), the incorporation of stable functional
groups (piperazine center)26 into the amide, as well as the
replacement of the benzamide/nicotinamide with an isoindolin-
1,3-dione (phthalimide) to produce a more rigid and in vivo
stable molecule.27 Studies demonstrated 5 to have a 3-fold
higher tumor uptake than 2, with rapid whole body clearance;27
however, further preclinical evaluation of [131I]5 found the
compound did not show the longitudinal in vivo stability
required to be a suitable therapeutic agent.28 This was
postulated to be due to instability of the phthalamide group
and provided the rationale for the work developed herein
utilizing the isoindolin-1-one moiety. We report the develop-
ment of a new class of melanin targeted agents, based on
systematic structural variations of 1−5, as potential [131I]
radiopharmaceuticals for treatment of malignant melanoma.
Presented is the chemical synthesis, [123I] radiolabeling, and
initial biological evaluation of these compounds.
Alternatively, synthesis of other analogues in the benzylpi-
perazine group (the 1,8-naphthyridine and partially reduced
1,8-naphthyridine moiety) proceeded via a convergent pathway
(Scheme 1, bottom). The 1,8-naphthyridine moiety was
chosen, as it is similar in structure to literature standard 4
except that the positions of the nitrogen atoms in the aromatic
ring are modified. The 1,8-naphthyridine moiety also gave easy
one-step access to the partially reduced moiety, readily
producing two compounds with different lipophilicity and π-
stacking characteristics for comparison. 1,8-Naphthyridine-2-
carboxylic acid (16) coupled to 1-Boc-piperazine (17), which
allowed access to the key Boc-protected intermediate 18, then
was reduced with Pd/C under H2(g) atmosphere to yield 19.
Subsequent Boc-deprotection of 18 and 19 with trifluoroacetic
acid (TFA) (isolated as the free amine) followed by N-
alkylation with 4-bromo or 4-iodobenzyl bromide afforded
iodinated standards and brominated compounds 20−21, and
23−24, respectively (Scheme 1, bottom).
RESULTS AND DISCUSSION
■
Chemistry. In an effort to develop melanin specific
radiotracers with improved in vivo stability, tumor uptake,
and clearance from radiosensitive organs, we have pursued two
In the second group of compounds (isoindolin-1-one
heterocycles, Figure 2b), the isoindolin-1-one (of 13−15)
B
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a
Scheme 1. Synthesis of Iodo-, Bromo-, and Stannylbenzylpiperazine Derivatives
a
Reagents and conditions: (A) HOBt, EDC, DIPEA, DMF, 16 h, rt; (B) TFA, CH2Cl2, 16 h, rt; C) 4-iodo or 4-bromobenzyl bromide, K2CO3,
CH3CN, 16 h, reflux; (D) Sn2(CH3)6, Pd(PPh3)4, toluene, 16 h, 80 °C; (E) Pd/C, H2(g), EtOH, 16 h, rt.
and benzyl moiety of the first group were retained; however,
the piperazine central linker was modified with four
heterocyclic amines. The four heterocycles chosen are all
structural modifications of the piperazine core, known for its
stability,27 to observe what effect this change to stability,
flexibility/rigidity would have on the in vivo behavior of the
molecules.
Scheme 2. Synthesis of Intermediates from the Isoindolin-1-
one Heterocyclic Group
a
The synthesis began with the corresponding Boc protected
intermediates (26, 31, 36), which were N-alkylated with either
4-iodo or 4-bromobenzyl bromide to give 27−28, 32−33, and
37−38 followed by Boc-deprotection with TFA and isolation as
the free amine as described in Scheme 2. The resulting amines
(29−30, 34−35, 39−40) were coupled with 2-(1-oxoisoindo-
lin-2-yl)acetic acid (12) to yield iodinated and brominated
compounds 41−42, 44−45, 47−48, respectively (Scheme 3).
The iodinated standard 53 and brominated compound 54 were
synthesized via an alternative method, as 4-amino-1-Boc-
piperidine hydrochloride (50) was commercially available. 12
was first coupled to 50 to give 51, followed by Boc-
deprotection with TFA, which was isolated as the free amine
52, that was then N-alkylated with 4-iodo or 4-bromobenzyl
bromide to give compounds 53−54 (Scheme 3).
Brominated compounds (10, 14, 21, 24, 42, 45, 48, 54) and
the literature nonradioactive iodinated standard 4 were each
individually treated with hexamethylditin and a catalytic
amount of tetrakis(triphenylphosphine)palladium(0) (Pd-
(PPh3)4) to yield the stannyl radiolabeling precursors (11,
15, 22, 25, 43, 46, 49, 55 (Schemes 1 and 3) and literature 56
(Supporting Information) for use in electrophilic [123I]
radioiodination.
a
Reagents and conditions: (C) 4-iodo or 4-bromobenzyl bromide,
K2CO3, CH3CN, 16 h, reflux; (B) TFA, CH2Cl2, 16 h, rt.
1H NMR spectra of compounds in the isoindolin-1-one
group were generally more complicated than their simple
benzylpiperazine analogues, predominantly because of either
conformational restriction or “potential” conformational
flexibility in concert with amide isomerism. Despite the
complex NMR spectra, complete characterization was possible,
details of which and associated spectra can be found in the
Supporting Information (Figure S1).
C
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a
Scheme 3. Synthesis of Compounds Containing the Heterocyclic Isoindolin-1-one Group
a
Reagents and conditions: (A) 12, HOBt, EDC, DIPEA, DMF, 16 h, rt; (B) TFA, CH2Cl2, 16 h, rt; (C) 4-iodo or 4-bromobenzyl bromide, K2CO3,
CH3CN, 16 h, reflux; (D) Sn2(CH3)6, Pd(PPh3)4, toluene, 16 h, 80 °C.
Table 1. [123I] Radiolabeling and Purification Conditions
a
radiotracer
precursor
HPLC mobile phase (CH3CN/H2O/modifier)
bd
retention time (min)
radiochemical yield (%)
,
[
[
[
[
[
[
[
[
[
123I]9
11
15
22
25
43
46
49
55
56
50:40:10
50:40:10
50:40:10
25:65:10
55:35:10
55:35:10
50:40:10
30:60:10
50:40:10
16.0
17.3
12.2
16.6
16.7
14.5
15.9
14.8
77.0 6.2 (n = 3)
83.3 10.0 (n = 3)
75.5 3.5 (n = 2)
68.8 11.9 (n = 3)
76.7 2.1 (n = 3)
76.0 4.6 (n = 3)
89.5 2.1 (n = 3)
58.7 5.9 (n = 3)
73.3 3.8 (n = 3)
bd
,
123I]13
123I]20
123I]23
123I]41
123I]44
123I]47
123I]53
123I]4
de
,
c,e
bd
,
bd
,
bd
,
de
,
bc
,
15.7
a
b
c
d
Flow at 2 mL/min. 0.1 M NH4HCO3, pH 8. Waters Xterra 10 μm, 300 mm × 100 mm. Phenomenex Bondclone 10 μm, 300 mm × 7.8 mm.
e
1% trifluoroacetic acid.
Radiochemistry. Radioiodination was achieved by reacting
the stannyl precursor with [123I]Na via standard electrophilic
iododestannylation chemistry in the presence of chloramine-T
as the oxidant to afford the corresponding [123I] analogues. The
select the most promising radiotracers for further in vivo
evaluation. Selection criteria included high and prolonged
tumor uptake over time (expressed as the remained radio-
activity (dpm) with decay correction) and high tumor-to-
background (lungs) ratio (Table S1) indicating fast clearance
from nontarget organs, in comparison to [123I]4. An example of
a typical SPECT planar image of the distribution in this study is
shown in Figure 3 ([123I]41), and the entire range of SPECT
images is available in the Supporting Information (Figure S2).
Several radiotracers experienced high thyroid uptake (in vivo
deiodination) including [123I]44 and [123I]47 at 72 h and [123I]
9 from 24 h onward. [123I]4 displayed significant thyroid uptake
visible from 3 h postinjection and also appeared to enter the
brain from early time points scans (prior to 1 h). Over the
study time period, unlike [123I]4, no localization of radioactivity
was observed in the brain for the eight new radiotracers,
indicating that they did not cross the blood−brain barrier. For
animals involved in the study of [123I]20, the scans at longer
time points (48−72 h) could not be performed because of the
ethical restrictions on the tumor growth and permissible size.
[
123I] labeled radiotracers were partially purified by solid phase
extraction using a C18 Sep-Pak Light cartridge prior to reverse
phase (RP)-HPLC purification to yield [123I] labeled radio-
tracers for all biological evaluations of radiochemical purity of
>95%. Radiotracers were labeled in moderate-to-high radio-
chemical yields (60−90%) (Table 1). Specific activity was
estimated to be >2 GBq/nmol based on the limit of detection
of the HPLC system.
SPECT Imaging Studies. The eight new radiotracers
([123I]9, [123I]13, [123I]20, [123I]23, [123I]41, [123I]44, [123I]47,
and [123I]53) as well as the literature radiotracer [123I]4 were
subjected to in vivo whole-body distribution monitoring over
72 h using SPECT imaging in C57BL/6J mice bearing the
B16F0 murine melanoma tumor. This time frame was chosen
to evaluate the potential [131I] therapeutic application of the
radiotracers. The SPECT imaging studies were performed to
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Figure 3. Representative planar SPECT image of the distribution of
[
123I]41 in C57BL/6J black mouse bearing B16F0 murine melanoma
tumor.
For all new radiotracers tested, radioactive signal was
observed in the eyes, which supported the specific uptake in
melanin-rich tissues; however, the study was conducted in
C57BL/6J mice that have high melanin pigmentation in the
eyes27,29 (as well as skin, hair, and spleen).30−33 Because of the
large differences between murine and human ocular geometry
as well as uveal melanin content, radioactivity uptake in the
eyes may not be an issue for clinical transfer.19 Additionally, the
first in human study of a similar benzamide-type radiotracer
developed by our research group ([18F]MEL050)34,35 indicated
that the radiotracer is safe with suitable biodistribution for PET
imaging.36
Figure 4. Comparison of (a) tumor-to-background (TBR) uptake
ratios and (b) tumor kinetic profiles over time of [123I] labeled
compounds in C57BL/6J black mice bearing B16F0 murine melanoma
tumor. Full tabulated data can be found in the Supporting Information
(Table S1). For animals involved in the study of [123I]20, the scans at
longer time points (48−72 h) could not be performed because of the
ethical restrictions on the tumor growth and permissible size.
Figure 4a shows the tumor-to-background ratios (TBRs) of
all studied radiotracers. For the period of 24−72 h all
radiotracers (except [123I]9) clearly possessed higher uptake
ratios compared to that of [123I]4. [123I]13, [123I]44, and [123I]
47 displayed a fast tumor wash-out compared to the remaining
new radiotracers; however, the TBRs were still greater than
Biodistribution Studies. Radiotracers [123I]20, [123I]23,
[
123I]41, and [123I]53 were injected into B16F0 tumor bearing
C57BL/6J mice to evaluate the potential of their corresponding
[
123I]4 at 72 h.
[
131I] labeled analogues as melanotic tumor therapeutics.
Figure 4b demonstrates the tumor kinetic profiles of the
Criteria to assess their potential therapeutic properties included
high and prolonged tumor uptake (indicating high affinity and
specificity for melanotic tissues), high tumor-to-radiosensitive
organ areas under the curves ratios (AUCRs) (indicating high
cumulative uptake in the tumor, yet low cumulative uptake in
radiosensitive organs), low thyroid uptake (indicating low in
vivo deiodination), and the amount of intact radiotracer in the
tumor (indicating in vivo stability).
Postinjection time points at 1, 3, 6, 16, 24, and 48 h were
selected to determine the distribution of each compound in
various tissues. Longer time points of 16, 24, and 48 h were
included to determine the long-term retention of the
radiotracers in the body. The values of tumor and
representative organ radioactivity concentration, expressed in
percent of injected dose per gram (% ID/g), are presented in
Table 2 and additional values for all organs measured are
detailed in the Supporting Information (Table S2).
studied radiotracers which are expressed as decay-corrected
radioactivity retained in the tumor over time. Tumor kinetic
profiles were utilized, as they are more representative in terms
of indicating the dynamic process of the radiotracer uptake into
the target and its clearance from the body or from nontarget
tissues over time. In comparison, for example, tumor-uptake
profiles present the static uptake ratios (tumor-to-organ) only
at a particular time point. At time point 24 h, [123I]13, [123I]20
(see Figure 4 caption about [123I]20), [123I]23, [123I]41, and
[
123I]53 had higher tumor uptake compared to [123I]4;
however, at 48 h only [123I]41 and [123I]53 demonstrated
higher tumor uptake than [123I]4. At 72 h only [123I]53
displayed higher tumor uptake than [123I]4. Although SPECT
imaging could not be obtained for [123I]20 after 24 h because of
ethical restrictions, the radiotracer initially showed great
potential and may have continued to if the trend (Figure 4)
remained as anticipated. On the basis of these results, the most
favorable radiotracers ([123I]20, [123I]23, [123I]41, and [123I]53)
were selected for further in vivo biological investigation
(biodistribution and metabolite studies) for evaluation as
potential [131I] therapeutic agents.
High uptake in the liver was observed for all four radiotracers
(16−42% at 1 h, [123I]41 > [123I]23 > [123I]20 > [123I]53),
suggesting an involvement of the clearance via the hepatic
portal pathway. By 16 h, the liver uptake of all radiotracers was
reduced to background level. The estimated biological half-lives
E
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a
Table 2. Distribution (% ID/g Organ SEM, n = 5) in Selected Organs of [123I]20, 23, 41, and 53 in C57BL/6J Mice Grafted
with F0 Melanoma Tumor (Decay Corrected)
b
compd
time (h)
tumor
eyes
large GIT
small GIT
thyroid
spleen
liver
kidney
[
123I]20
123I]23
123I]41
123I]53
1
17.1 2.8
20.0 2.8
19.4 2.6
18.4 1.1
14.4 1.0
13.1 3.9
19.2 1.0
32.1 2.3
32.5 1.4
20.4 2.6
12.9 1.4
5.6 0.9
32.0 1.2
36.0 2.4
37.2 1.7
32.9 2.4
35.1 3.4
31.6 1.2
40.3 2.8
56.4 5.4
52.8 5.8
28.6 1.3
30.8 1.4
19.0 1.1
18.2 4.1
18.5 4.7
21.8 2.0
22.1 1.4
18.4 0.9
15.8 0.7
13.7 0.5
20.8 0.6
28.1 2.5
40.4 3.0
41.4 2.3
38.7 3.1
3.2 0.2
11.9 1.6
18.6 2.1
1.8 0.1
1.4 0.2
0.3 0.02
7.9 0.4
59.6 10.4
87.1 3.9
2.3 0.2
1.7 0.3
0.2 0.04
3.5 0.8
21.4 2.3
23.1 2.3
1.4 0.2
1.0 0.1
0.1 0.01
6.6 0.3
10.9 0.4
17.6 1.0
8.1 0.9
8.0 1.2
0.6 0.05
14.4 0.8
14.1 0.9
6.1 0.9
1.3 0.1
0.9 0.05
0.2 0.02
45.6 3.9
63.8 4.2
11.5 0.7
1.4 0.1
0.8 0.1
0.1 0.02
21.4 4.9
15.2 0.6
2.6 0.2
0.5 0.04
0.3 0.04
0.1 0.004
11.9 0.9
12.0 0.5
9.8 0.7
3.7 0.3
3.8 0.3
0.3 0.03
2.3 0.1
2.2 0.5
3.5 0.5
1.6 0.2
2.9 1.3
5.3 0.9
5.1 0.4
2.7 0.1
2.7 0.5
2.5 0.5
2.0 0.4
2.1 0.4
1.9 0.5
1.5 0.2
2.8 0.4
1.0 0.4
1.0 0.3
3.4 1.7
3.4 0.2
3.4 0.1
3.1 0.2
2.3 0.2
2.6 0.3
2.6−8.6
2.3−13.3
2.1−16.7
0.8−3.4
0.5−3.4
0.2−4.9
3.5−24.4
2.0−7.3
1.0−1.9
0.3−4.0
0.2−2.4
0.05−0.3
0.2−9.8
0.5−5.6
0.4−4.1
0.2−5.1
0.05−3.4
0.09−3.0
9.6−12.6
7.6−16.9
3.6−17.7
1.3−17.3
1.2−17.8
0.1−2.0
15.9 0.8
13.4 0.8
10.1 0.3
2.4 0.2
1.8 0.1
0.7 0.04
19.5 0.7
13.2 0.5
6.3 0.6
0.9 0.1
0.8 0.05
0.2 0.01
12.3 0.8
5.6 0.2
3.1 0.1
0.7 0.05
0.4 0.04
0.2 0.01
41.8 1.0
38.8 1.1
35.1 3.0
7.1 0.3
7.8 0.6
1.9 0.2
7.5 0.5
6.2 0.4
5.2 0.4
1.6 0.1
1.4 0.02
0.5 0.04
15.6 1.3
9.0 0.6
10.0 5.1
1.2 0.1
1.0 0.1
0.4 0.02
15.8 3.6
8.4 0.2
4.1 0.2
1.0 0.1
0.5 0.1
0.2 0.01
8.0 0.5
7.4 0.2
5.0 0.7
1.8 0.1
1.9 0.2
0.3 0.03
3
6
16
24
48
1
[
[
[
3
6
16
24
48
1
10.3 2.5
10.8 1.6
10.4 0.9
11.6 1.6
7.8 1.6
3
6
16
24
48
1
5.0 0.7
6.2 0.8
3
11.6 0.8
18.1 10.0
18.1 2.0
15.2 3.8
13.2 4.3
6
16
24
48
2.4 0.3
a
b
Table of organ distribution in all organs can be found in Supporting Information, Table S2. % ID/g in spleen are presented as range of values
because of differences in the extent of pigmentation of the spleen.10
were based on the biodistribution profile of the four tracers in
the liver and kidney, as the main route of metabolism and
excretion, respectively. The estimated biological half-lives for
[
[
part or all of the spleen is heavily pigmented and can also
display interindividual variation.30,33
Whole body clearance (>98% of injected activity) was
observed for each tracer at 16 h postinjection (Figure 5). These
123I]41 and [123I]23 were 3−6 h and 6−16 h for [123I]20 and
123I]53. The kidneys displayed low-to-medium uptake,
∼16% ID/g at 1 h for [123I]23 and [123I]41 and ∼8% ID/g
at 1 h for [123I]20 and [123I]53, all of which decreased to
background level by 16 h. Thyroid uptake was low for all
radiotracers and generally plateaued at ∼3% ID/g across all
time points, indicating minor amounts of in vivo deiodination.
Generally, uptake in the GIT (small and large) was low for all
radiotracers, peaking at 3−6 h (∼17% ID/g) and decreasing to
background levels over time except for [123I]23 which initially
showed very high uptake (large GIT 87% ID/g at 6 h and small
GIT 64% ID/g at 3 h) and then continued to decrease to
background level over time.
For all radiotracers tested, high radioactivity uptake was
observed in melanin containing tissues (i.e., tumor, eyes, and
spleen). In the tumor, [123I]23 exhibited the highest uptake
(33% ID/g at 6 h) but significantly decreased with time,
whereas high and prolonged tumor uptake was observed for
Figure 5. Activity remaining in the body (% ID/g SEM n = 5, decay
corrected) at various time points postinjection of the four [123I]
radiotracers in C57BL/6J black mice with B16F0 tumors. Data were
obtained from the biodistribution studies.
[
123I]20 and [123I]53 (13% ID/g at 48 h). High and prolonged
eye uptake was observed for all radiotracers (at least 16% ID/g
at 48 h); again [123I]23 exhibited the highest uptake (56% ID/g
at 3 h) and then significantly decreased with time. The spleen
displayed a varied range of radioactive uptake for all
radiotracers tested, from background level to a peak at
24% ID/g ([123I]20 at 1 h). This result was not unexpected
because of large differences in the extent of pigmentation of the
spleen (splenic melanosis) found in black mice, particularly the
C57BL/6J strain.31,32 This is a normal condition that can be
found in various organs but most commonly the spleen where
data re-enforced the findings from the initial SPECT studies
which supported a melanin-related uptake mechanism with a
high selectivity and specificity for melanin content.
In order to evaluate the therapeutic potential of these
radiotracers, their nonstochastic effects have to be considered.
The therapeutic potential of a radiotracer is indicated by a high
cumulative uptake in the tumor (tumor AUCs), a low
F
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Journal of Medicinal Chemistry
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a
Table 3. Area under the Curve (AUC) of Lead [123]I Labeled Compounds in Radiosensitive Organs
radiosensitive organ AUC (% ID g−1 h−1
)
compd
tumor
skin
eyes
bone
GIT
thyroid
[
123I]20
123I]23
123I]41
123I]53
746.8 61.9
768.1 24.9
716.7 107.7
396.0 27.0*
16.6 0.4
24.4 7.5
32.7 4.8
18.2 2.8
1601.4 72.0
1501.4 73.1
1737.4 53.4
904.9 28.3*
19.0 4.1*
12.3 1.3
29.0 2.0
136.5 8.0*
428.8 15.3
246.2 11.4
135.8 7.3*
154.3 18.1
107.6 5.7
122.2 8.0
90.4 25.9
[
[
[
**
6.1 0.3
a
Data are derived from the biodistribution studies and presented as the mean of the ratios SEM, n = 5. Areas under the curves are calculated using
*
**
GraphPad Prism, version 5.04. One-way ANOVA (radiotracers as factor), followed by Bonferroni post hoc tests. 0.01 < P < 0 0.05. P < 0.001.
a
Table 4. Tumor-to-Radiosensitive Organ Area under the Curve Ratios (AUCRs) of Lead [123]I Labeled Compounds
tumor-to-radiosensitive organ AUC ratio
compd
tumor/skin
tumor/eyes
tumor/bone
tumor/GIT
**
tumor/thyroid
[
123I]20
123I]23
123I]41
123I]53
45.3 4.2
40.9 8.1
23.1 4.5
24.4 5.2
0.47 0.03
0.52 0.02
0.42 0.07
0.44 0.04
44.0 7.0
65.5 8.2
5.5 0.4
5.0 0.5
7.2 0.3
6.0 1.1
5.8 1.4
[
[
[
1.8 0.03
2.9 0.4
3.0 0.4
***
25.0 3.8
65.8 5.2
**
a
***
One-way ANOVA (radiotracers as factor), followed by Bonferroni post hoc tests. P < 0.001.
P < 0.01.
cumulative uptake in the radiosensitive organs (radiosensitive
organ AUCs, Table 3), and a high ratio of the two uptakes
(tumor-to-radiosensitive organ AUCRs, Table 4). Radio-
sensitive organs such as the gastrointestinal tract (stomach,
small and large intestines), skin, eyes, bone marrow, and
thyroid18 were selected for calculation of tumor-to-organ
cumulative uptake (% ID/g) over time ratios (AUCRs) and
served as a direct indication of the potential absorbed dose of
the radiotracer to the radiosensitive organs and indirectly their
therapeutic potential (Table 4).
point of 24 h is particularly applicable to these radiotracers for
their therapeutic potentials. In the tumor and eyes (melanin
containing tissues), ∼90% of the radioactive signal corre-
sponded to intact tracers [123I]41 and [123I]53 (at 3 h) and
showed prolonged stability over time (∼80% at 24 h).
Conversely, [123I]20 and [123I]23 showed poor in vivo stability
in the tumor and eyes (∼60% at 3 h, ∼35% at 24 h). All tracers
exhibited fast breakdown in the urine and plasma (mostly
undetectable). These results demonstrated that once bound to
melanin in pigmented tissues [123I]41 and [123I]53 remained
highly unchanged. The extraction efficiencies for all tracers in
all tissues and time points tested were found to be diverse
(∼60−90%) (Supporting Information Table S4) but are in
accordance with other compounds of similar structure (5).25
Statistical analysis of AUCRs was performed for each
radiosensitive organ and each radiotracer. One-way ANOVA
analysis of the time−activity areas under the curves (AUCs) for
each radiosensitive organ among the radiotracers, with
radiotracers as an independent factor, showed that the
cumulative uptake in the tumor over time of [123I]20, [123I]
23, and [123I]41 are significantly higher than that of [123I]53
(F3,16 = 7.319, P = 0.0026). However, there is no significant
difference among these three radiotracers. [123I]53 showed
lowest cumulative uptake over time in the eyes (0.01 < P <
0.05), bone (P < 0.001), and GIT (0.01 < P < 0.05) compared
to other radiotracers. Separate one-way ANOVA analyses for
each of the tumor-to-radiosensitive organ AUCRs among the
radiotracers indicated that there were no statistically significant
difference between the tumor-to-skin, tumor-to-eyes, and
tumor-to-thyroid AUCRs. However, there was an overall
significant difference among the tumor-to-GIT AUCRs (F3,16
= 20.92, P < 0.0001) and tumor-to-bone AUCRs (F3,16 = 9.768,
P = 0.0007) for these radiotracers. Bonferroni post hoc analysis
indicated that [123I]23 had the highest tumor-to-GIT AUCR
(i.e., lowest absorbed dose to the GIT, P < 0.001), and both
CONCLUSIONS
■
With the aim of developing new radiotherapeuticals for use in
metastatic melanoma, we have synthesized iodinated melanin
targeting compounds containing either a benzylpiperazine or an
isoindolin-2-one heterocyclic moiety. Eight new compounds
and literature compound 4 were radiolabeled with [123I] and
whole-body-imaged in B16F0 tumor bearing C57BL/6J mice
using SPECT. All compounds displayed high and distinct
uptake between targets and nontargets and good tumor-to-
background ratio, compared with literature compound [123I]4.
The most favorable compounds from the SPECT study
([123I]20, [123I]23, [123I]41, and [123I]53) were further
investigated by biodistribution and metabolite studies for
their therapeutic potential against melanoma. [123I]20 and
[
123I]53 displayed the favorable pharmacokinetic properties in
123I]23 and [123I]53 had the highest tumor-to-bone AUCRs
the biodistribution studies with clear delineation of the tumor
as early as 1 h postinjection and prolonged tumor retention.
Although [123I]53 appeared to have the lowest cumulative
uptake in the tumor over time compared to other radiotracers,
[
(i.e., lowest absorbed dose to bone, P < 0.01) compared to
other radiotracers.
Metabolite Studies. The in vivo stability of [123I]20, [123I]
23, [123I]41, and [123I]53 were evaluated by radio-TLC and
radio-HPLC analysis at 3 and 24 h postinjection in the tumor,
eyes, spleen, urine, and plasma (Supporting Information Table
S3). Overall, both analytical methods examined the retained
radioactivity that was bound to a tissue corresponding to the
intact radiotracer and yielded results in concordance with each
other presenting similar trends of in vivo stability. The time
[
123I]20 had the lowest absorbed dose to GIT; however, it had
low stability. Both [123I]23 and [123I]53 had the lowest
absorbed dose to bone. Metabolite analysis of [123I]41 and
[
123I]53 displayed high and prolonged amounts of intact tracer
in melanin containing tissues. Results herein suggest that
compound [123I]53 is an ideal candidate from this new series to
proceed with further preclinical [131I] therapeutic evaluation.
G
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dried over anhydrous Na2SO4, filtered, and evaporated under reduced
pressure. The crude residue was purified by column chromatography
(silica gel, 0−15% CH3OH/EtOAc), and the products were obtained
as either a pale yellow oil or waxy solid. Full compound details are
available in the Supporting Information.
General Procedure B: Amine Deprotection. The desired Boc
protected amine (∼1 g) was stirred in TFA/CH2Cl2 (1:4, 5 mL) for
16 h at rt. To isolate the product as the trifluoroacetate salt, the solvent
was removed and the residue dissolved in CH2Cl2. To isolate as the
free amine, the solution was first adjusted to pH 12 (with aqueous 1 M
NaOH) before concentration and redissolution in CH2Cl2. It was then
washed with brine (2 × 15 mL) and H2O (2 × 15 mL). The organic
phase was dried over anhydrous Na2SO4, filtered, and evaporated to
dryness to obtain the deprotected product. Full compound details are
available in the Supporting Information.
EXPERIMENTAL SECTION
■
Materials for Chemical Synthesis and Radiochemistry.
Paraformaldehyde, N-benzylmaleimide, 1-chloroethyl chloroformate,
homopiperazine, 1-Boc-piperazine (17), 4-iodobenzyl bromide, 4-
bromobenzyl bromide, 1-(4-bromobenzyl)piperazine (7), 4-amino-1-
Boc-piperidine hydrochloride (50), nicotinic acid (8), 2-(1-oxoisoin-
dolin-2-yl)acetic acid (12), methyl pyruvate, 2-amino-3-pyridine
carboxaldehyde, 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline hydro-
chloride, benzyl bromoacetate, 2-carboxybenzaldehyde, glycine methyl
ester hydrochloride, hexamethylditin, potassium iodide, Pd(PPh3)4,
TFA, and chloramine-T hydrate (98%) were purchased from Sigma-
Aldrich. Di-tert-butyl dicarbonate was purchased from Alfa Aesar. 1-(4-
Iodobenzyl)piperazine (6) was purchased from ACB Blocks. All
HPLC solvents were of HPLC grade and were filtered before use. All
other reagents were of analytical grade or higher and used as received.
Na123I no-carrier added was purchased as from ANSTO Health. Silica
gel TLC plates with a preconcentration zone used in metabolite
studies were purchased from Merck.
General Procedure C: N-Alkylation. The appropriate amine (1−
10 g, 1.0 equiv) was dissolved in CH3CN (10−100 mL), followed by
addition of 4-iodobenzyl bromide or 4-bromobenzyl bromide (1.1
equiv) and K2CO3 (2.0 equiv) and the solution heated to reflux for 16
h. The solution was filtered, concentrated and the residue purified by
column chromatography (silica gel, petroleum spirit/EtOAc, 50:50).
Full compound details are available in the Supporting Information.
General Procedure D: Synthesis of Stannanes. The desired
brominated compound (10, 14, 21, 24, 42, 45, 48, 54) or literature
iodinated 4 (∼ 100 mg) was dissolved in toluene (∼20 mL), and while
under nitrogen atmosphere, hexamethylditin (1.1 equiv) and Pd-
(PPh3)4 (0.1 equiv) were added and the mixture was heated to 80 °C
for 16 h. The reaction was monitored by TLC (either petroleum
spirit/EtOAc solutions or EtOAc/CH3OH solutions). The reaction
mixture was filtered over Celite and evaporated to dryness. The crude
material was purified by silica gel chromatography (either petroleum
spirit/EtOAc solutions or EtOAc/CH3OH solutions), followed by
preparative HPLC. Full details for stannyl radiolabeling precursors
(11, 15, 22, 25, 43, 46, 49, 55, and 56) are available in the Supporting
Information.
Radiochemistry. An ethanolic solution (100 μL of 1 mg/mL) of
the stannyl precursor (11, 15, 22, 25, 43, 46, 49, and 55 and 56) was
treated with a solution of Na123I (370−555 MBq) followed by
chloramine-T (100 μL, 1 mg/mL H2O) and 1 M HCl (100 μL). After
5 min at room temperature (rt) the solution was quenched with
Na2S2O5 (100 uL, 50 mg/mL H2O) and neutralized with NaHCO3
(100 μL, 50 mg/mL H2O). The crude reaction mixture was diluted
with H2O (10 mL) and loaded onto an activated Waters Sep-Pak C18
Light cartridge (cartridges were activated with ethanol (2 mL) and
then washed with water (15 mL) before use). The cartridge was eluted
with CH3CN (∼0.7 mL) and the eluate diluted with H2O (∼0.7 mL)
and purified on a semipreparative C18 RP HPLC column using
conditions described in Table 1. HPLC purification was performed
using a Waters Empower 2 system using a Waters 600 pump, Waters
in-line degasser AF, Waters 2489 UV/visible detector measured at 254
nm, Carroll & Ramsey radioactivity detector, and a 7725i Rheodyne
injector valve. The product was collected, diluted with H2O (10 mL),
and loaded onto an activated Waters Sep-Pak C18 Light cartridge. The
cartridge was eluted with EtOH (1 mL) and the solvent dried in vacuo
and then formulated in saline solution with <5% ethanol for biological
evaluation. In this formulation, tracers were found to be stable with
purity of >95% up to 3 h.
Instrumentation. Nuclear magnetic resonance (NMR) spectra
were recorded using a Bruker Avance DPX-400 spectrometer
1
operating at 400.13 MHz for H NMR and at 100.61 MHz for 13C
NMR. Low-resolution mass spectrometry (LR-MS) was performed on
a Micromass ZQ quadrupole mass spectrometer, and high-resolution
mass spectrometry (HR-MS) was performed at the University of
Wollongong, Australia, using a Bruker Daltonics BioApex-II 7T
FTICR spectrometer equipped with an off-axis analytical electron
spray ionization source.
All nonradioactive standards and radiolabeling precursors were
purified by HPLC using a Waters XBridgeC18 100 mm × 30 mm
column (unless specified otherwise), with flow rate at 20 mL/min
under gradient conditions of (A) CH3CN, (B) H2O, (C) 100 mM pH
8 NH4HCO3. Detection wavelength was either 210 or 254 nm. Two
systems were used for purification, either Waters PrepLC pump
coupled with 717 autosampler and 486 UV detector or Waters 600
controller pump coupled with 717 autosampler and 996 photodiode
array UV detector.
HPLC purity analysis was performed using a Waters Empower 2
system with a Waters 600 pump, Waters in-line degasser AF, Waters
temperature control module II, Waters 717 autosampler, and Water
2996 PDA. Either an Alltech Alltima C18 (150 mm × 4.6 mm, 5 μm)
or Waters XTerra RP (150 mm × 4.6 mm, 5 μm) analytical column
was used, with absorbance measured at 210 or 254 nm. Samples were
prepared as 1 mg/mL, with a 10 μL injection. Solvent conditions were
as shown in Table 5.
Table 5. HPLC Solvent Conditions
% A
(CH3CN
or
% C (100 mM
NH4HCO3, pH 8, or
1% TFA/99% H2O)
flow
% B
entry time (mL/min) CH3OH) (H2O)
1
2
3
0
20
21
1.00
1.00
1.00
10
90
10
80
0
10
10
10
80
Percentage purity of the iodinated standards and stannyl precursors
were calculated from the area under the peak using Empower software
(Waters). Preparative HPLC was conducted on nonradioactive
iodinated standards and stannyl radiolabeling precursors to achieve
purity of >95%.
General Procedure A: Amide Coupling. To a solution of the
appropriate amine (1.0 equiv) in DMF (10 mL) were added 1-
hydroxybenzotriazole hydrate (HOBt) (1.2 equiv), 1-(3-dimethylami-
nopropyl)-3-ethylcarbodiimide hydrochloride (EDC) (1.2 equiv), and
diisopropylethylamine (DIPEA) (3 equiv), followed by the acid of
interest (1.1 equiv, either purchased or synthesized). The reaction
mixture was stirred under nitrogen for 16 h at rt, diluted with ethyl
acetate (EtOAc) (50 mL), and washed with H2O, saturated
NaHCO3(aq), and brine (3 × 20 mL, each). The organic phase was
Animals. Animal experiments were performed according to the
Australian Code of Practice for the Care and Use of Animals for
Scientific Purposes and were approved by the ANSTO Animal Care
and Ethics Committee. Female C57BL/6J mice aged 5−6 weeks old
were obtained from the Animal Resource Centre (Perth, Australia).
The animals were kept at a constant temperature of 22 2 °C on a
12−12 h light−dark cycle with light on at 09:00 a.m. Food and water
were freely available.
Cell Culture and Tumor Induction. The B16F0 murine
melanotic melanoma cells were originally obtained from the European
Collection of Cell Cultures (ECACC, U.K.) and cultured in RPMI-
1640 supplemented with 10% fetal calf serum at 37 °C under 5%
CO2/95% atmosphere. Cells were grown to subconfluent monolayers
H
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before being detached using trypsin for use in animal models. After a
week of acclimatization, mice were subcutaneously inoculated on the
left flank with 3 × 105 B16F0 cells in 100 μL of Ca2+/Mg2+-free
phosphate buffered saline. The procedure was performed without
anesthetic. B16F0 tumors were allowed to develop for 11 days before
the animals were used in experimentation with ∼77−100% of the
animals having developed desirable-sized tumors. Tumors used in
experiments were 2−12 mm in diameter.
ANOVA analyses (radiotracers as independent factor) followed by
Bonferroni post hoc tests were employed to determine the level of
statistically significant variation in the tumor-to-radiosensitive organ
AUC ratios among the radiotracers. Significance was set at P ≤ 0.05.
Metabolite Studies. The amounts of intact [123I]20, [123I]23,
[
123I]41, and [123I]53 in the tumor, eyes, spleen, urine, and plasma, of
B16F0 tumor bearing C57BL/6J mice, were quantified by radio-HPLC
and radio-TLC analysis at 3 and 24 h after injection of 20 MBq of the
radiotracer. After the samples were initially collected, they were placed
in a γ counter (PerkinElmer 1480 Wallace Wizard 3) and the activity
(cpm) and time of measurement were recorded.
SPECT Imaging Studies. The whole-body distribution of [123I]9,
[
[
123I]13, [123I]20, [123I]23, [123I]41, [123I]44, [123I]47, [123I]53, and
123I]4 in B16F0 tumor bearing C57BL/6J mice was followed over 72
h using SPECT imaging. The procedure was performed using a high-
resolution γ-camera (X-SPECT; Gamma Medica Inc., USA) designed
for laboratory animals and equipped with an array of discrete 2 mm ×
2 mm × 6 mm NaI(Tl) crystals optically isolated from each other and
a high-resolution, parallel-hole collimator of 12.5 cm × 12.5 cm field of
view. Animals (n = 3, per tested compound) were injected with 18−20
MBq of radiotracer (in 100 μL volume) through the lateral vein before
being anesthetized via inhalant isoflurane (forthane, 5% induction, and
2−2.5% maintenance) in 200 mL/min oxygen delivered through a
nose-cone fitted to the animal bed. Body temperature was maintained
using a heating pad set at 37 °C and respiration rate monitored
throughout the scanning period using a pressure sensor (BioVet, m2m
Imaging Corp, USA). Mice were planar imaged at 10−60 min (5 min
frames), 3 and 6 h (10 min), 24 h (20 min), 48 h (40 min), 72 h (60
min) (tumor permitting) and were recovered from anesthetic with
access to food and water between scans.
SPECT images were analyzed by drawing a region of interest (ROI)
around the tumor, and the lung as a background region which
represents the mediastinal blood pool excluding regions of tracer
accumulation. The lung ROI was chosen as background for maximum
pixel value (MPV) calculation, as it results in a more conservative
estimation for the differences in tumor to nontumor comparisons due
to lower attenuation of the photon through the lung region which is
predominantly air. The MPV within the ROI was then divided by the
maximum pixel value within the background ROI to determine a
tumor to nontumor ratio. Further to this, corrections were made to the
MPV for radioactivity decay, including for scan length so that activity−
time course kinetic information on the radiotracer for the regions
could be determined.
Whole organ tissue samples (tumor, eyes, and spleen) were
suspended in CH3CN/water (3:2, 500 μL/50 mg tissue), 5 μL of the
corresponding nonradioactive iodine standard (1 mg/mL in CH3OH)
and 5 μL KI (1 mg/mL in water) were added, and the sample was
exposed to an ultrasonic probe (Ultrasonic Processor, Misonix Inc.,
Farmingdale, NY, USA) for 2 min before being centrifuged at 5000
rpm for 5 min. The supernatant was removed, and the radioactivity of
the precipitate was measured using a γ counter to quantify the
extraction efficiency. If necessary, a second extraction was performed
to ensure maximum recovery of the radioactivity. An appropriate
amount of the supernatant, based on the activity level (cpm), was
collected (∼100 μL) and diluted with water (up to 1.5 mL) for HPLC
analysis or evaporated to dryness on a rotary evaporator under vacuum
for TLC analysis.
To the urine (10 μL diluted to 1 mL with water) were added 5 μL
of the corresponding nonradioactive iodine standard (1 mg/mL in
CH3OH) and 5 μL KI (1 mg/mL in water) before being analyzed by
HPLC and TLC. For HPLC analysis, plasma (50 μL) was mixed with
5 μL of the corresponding nonradioactive iodine standard (1 mg/mL
in CH3OH) and 5 μL KI (1 mg/mL in water) before being analyzed
by HPLC. For TLC analysis, plasma (50 μL) was mixed with 5 μL of
the corresponding nonradioactive iodine standard (1 mg/mL in
CH3OH), 5 μL KI (1 mg/mL in water), and CH3CN (500 μL) before
being centrifuged (Heraeus Biofuge primoR centrifuge) at 5000 rpm
for 5 min; the supernatant was analyzed by TLC.
The TLC samples were diluted with CH3OH (25−50 μL) before
being applied to the concentrating zone of the silica TLC plate in
conjunction with the corresponding [123I] labeled and nonradioactive
iodine standard. Varied TLC solvent systems of EtOAc/CH3OH +
aqueous ammonia solution (200 mL) were utilized for the four
Biodistribution Studies. [123I]20, [123I]23, [123I]41, and [123I]53
(0.37−1.85 MBq, 100 μL) were injected intravenously into C57BL/6J
mice via the tail vein. Animals for time points of 1−24 h were injected
with approximately 0.37 MBq of the radiotracers, and 1.85 MBq was
injected for the 48 h time point. Postinjection points of 1, 3, 6, 16, 24,
and 48 h were chosen for determining the distribution of compounds
in tumors and in various organs and tissues. At the defined time point,
mice were sacrificed, selected organs were dissected, weighed, and the
radioactivity was measured with a Wallac 1480 γ-counter (Perki-
nElmer, MA, USA). The fraction of injected activity [percent injected
dose (% ID)] in the organ was calculated by comparison with suitable
dilutions of the injected dose. Then, the radioactivity concentration in
the organ (% ID/g) was ascertained by dividing the % ID for each
organ by the weight of the organ, assuming a uniform density of 1 g/
cm3. After decay correction, data were normalized with the individual
injected dose for each animal, the standard mouse body weighed (20
g), then corrected for the tail injected dose. The remaining activity in
the carcass was also determined in order to obtain the total activity in
the mouse at each time point.
[
123I]tracers analyzed: [123I]20 95:5 + 10 drops; [123I]23 90:10 + 10
drops; [123I]41 and [123I]53 85:15 + 10 drops. Visualizing the
retention of the nonradioactive standard spots was by a UV lamp,
while the radioactive spots were visualized using a phosphorimager
(Typhoon FLA 7000 Phophorimager, GE Healthcare, Rydalmere,
Australia, with Fuji Film Multigage 3.0 software). The intact tracer was
identified as the radioactive spot containing the identical Rf value
corresponding to the nonradioactive standard observed under the UV
lamp. Integration of the active spot in relation to all the activity in the
TLC lane gave the percentage of intact tracer.
Radio-HPLC for metabolite analysis was performed using a Waters
Empower 2 system using a two-stage pump system including a Waters
515 isocratic pump and a Waters 600 gradient pump, Waters in-line
degasser AF, Waters 2489 UV/visible detector measured at 254 nm,
IN/US Systems Posi-Ram detector, and a 7725i Rheodyne injector
valve. The separation followed the method of Hilton et al.20
A
precolumn (Waters Oasis HLB, 25 μm, 20 mm × 3.9 mm) and a
reverse phase HPLC column (Phenomenex Bondclone C18, 10 μm,
250 mm × 4.6 mm or Phenomenex Synergi Max-RP 80A C18, 4 μm,
250 mm × 4.6 mm) in series, with a switching valve between columns,
was utilized (Table 6). The precolumn was washed with 1% CH3CN
in water for 3 min at 1.5 mL/min, and then the solvent direction was
switched to include the HPLC column. Both columns in series were
then eluted over 10 min. The radioactivity peak corresponding to the
nonradioactive standard was compared to the total activity registered
in the radiochromatogram to give the fraction of unchanged ligand in
the sample.
Areas under the curves (AUCs) of the tumor and selected
radiosensitive organs were calculated using GraphPad Prism, version
5.04 (CA, USA). Tumor-to-radiosensitive organ AUC ratios (AUCRs)
were calculated by dividing the tumor AUC by a corresponding organ
AUC. Data from Table 3 are presented as the mean of percentage of
injected dose per gram of tissues (% ID/g) SEM, n = 5, and as the
mean of tumor-to-radiosensitive organ AUC ratios (AUCRs) SEM,
n = 5, which were derived from the biodistribution study.
Raw data were analyzed for significant outliers by Grubb tests, and
none were detected. Statistical analyses were performed using
GraphPad Prism, version 5.04 (CA, USA). Separate within one-way
I
DOI: 10.1021/acs.jmedchem.5b00777
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Pd(PPh3)4, tetrakis(triphenylphosphine)palladium(0); TLC,
thin layer chromatography; TFA, trifluoroacetic acid
Table 6. HPLC Chromatographic Conditions Used for
Metabolite Studies
chromatographic conditions
flow rate
REFERENCES
analysis
column
(CH3CN/modifier, v:v)
(mL/min)
■
a
[
[
[
[
123I]20
123I]23
123I]41
123I]53
Phenomenex
Synergi
50:50
1.0
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a
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b
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a
0.1 M NH4C2H3O2. 0.01 M NH4HCO3.
ASSOCIATED CONTENT
* Supporting Information
Full chemical characterization, extended NMR discussion,
entire SPECT images, full biodistribution, metabolite data,
and a csv file containing molecular formula strings. The
Supporting Information is available free of charge on the ACS
Publications website at DOI: 10.1021/acs.jmedchem.5b00777.
■
S
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AUTHOR INFORMATION
Corresponding Author
*E-mail: Ivan.Greguric@ansto.gov.au. Phone: +(61) 2 9717
3759. Fax: +(61) 2 9717 9262.
■
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§M.P.R. and V.N. contributed equally.
́
Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge the Cooperative Research Centre
for Biomedical Imaging Development (CRCBID) for financial
support. We thank Xiang Liu and Branko Dikic for chemical
synthesis. We thank Kerynne Belbin, Geetanjali Dhand, and
Emma Millard for assistance with animal studies and Iveta
Kurlapski for chemical and radiochemical technical assistance.
ABBREVIATIONS USED
■
CH3CN, acetonitrile; AUC, areas under the curve; AUCR, ratio
of areas under the curve; CH2Cl2, dichloromethane; dpm,
disintegrations per minute; DIPEA, N,N-diisopropylethyl-
amine; DMF, dimethylformamide; equiv, equivalent; EtOH,
ethanol; EtOAc, ethyl acetate; EDC, 1-ethyl-3(3-
dimethylaminopropyl)carbodiimide; HPLC, high performance
liquid chromatography; HR-MS, high-resolution mass spec-
trometry; h, hour; HOBt, hydroxybenzotriazole; NMR, nuclear
magnetic resonance; % ID/g, percent of injected dose per
gram; 123I, iodine-123; 131I, iodine-131; LR-MS, low-resolution
mass spectrometry; RP, reverse phase; rt, room temperature;
SPECT, single photon emission computed tomography;
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