Positron Emission Tomography Imaging of Neurotensin Receptor-Positive Tumors with68Ga-Labeled Antagonists: The Chelate Makes the Difference Again

Article abstract of DOI:10.1021/acs.jmedchem.1c00523

Neurotensin receptor 1 (NTS₁) is involved in the development and progression of numerous cancers, which makes it an interesting target for the development of diagnostic and therapeutic agents. A small molecule NTS₁antagonist, named [

Full text of DOI:10.1021/acs.jmedchem.1c00523

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Positron Emission Tomography Imaging of Neurotensin Receptor-
68
Positive Tumors with Ga-Labeled Antagonists: The Chelate Makes
the Difference Again
Emma Renard, Mathieu Moreau, Pierre-Simon Bellaye, Mélanie Guillemin, Bertrand Collin,
Aurélie Prignon, Franck Denat, and Victor Goncalves*
Cite This: J. Med. Chem. 2021, 64, 8564−8578
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ABSTRACT: Neurotensin receptor 1 (NTS1) is involved in the
development and progression of numerous cancers, which makes it an
interesting target for the development of diagnostic and therapeutic
agents. A small molecule NTS1 antagonist, named [177Lu]Lu-
IPN01087, is currently evaluated in phase I/II clinical trials for the
targeted therapy of neurotensin receptor-positive cancers. In this study,
we synthesized seven compounds based on the structure of NTS1
antagonists, bearing different chelating agents, and radiolabeled them
with gallium-68 for PET imaging. These compounds were evaluated in
vitro and in vivo in mice bearing a HT-29 xenograft. The compound
[68Ga]Ga-bisNODAGA-16 showed a promising biodistribution profile
with mainly signal in tumor (4.917 0.776%ID/g, 2 h post-injection).
Its rapid clearance from healthy tissues led to high tumor-to-organ ratios, resulting in highly contrasted PET images. These results
were confirmed on subcutaneous xenografts of AsPC-1 tumor cells, a model of NTS1-positive human pancreatic adenocarcinoma.
years of optimization, they identified SR142948A, a drug
candidate with nanomolar affinity and potency for neurotensin
receptors.19,20 Starting from the structure of SR142948A, a few
radiotracers were developed for the diagnosis or therapy of
neurotensin receptor-positive cancers. Such tracers present
several advantages: in the absence of activation of receptor’s
signaling pathways, they produce no pharmacological effect;
they often show better metabolic stability than peptides,21,22 and
their small size allows for a rapid biodistribution, with fast renal
clearance.
Furthermore, it has been shown that agonists and antagonists
may not necessarily recognize the same conformational states of
their receptors.23−25 This phenomenon has also been observed
with neurotensin receptors, by Betancur et al., who showed that
the number of receptors labeled by the antagonist [3H]-
SR142948A on rat brain membranes was 80% greater than that
detected when using the agonist [3H]neurotensin.20
INTRODUCTION
■
Molecular imaging is a non-invasive technique that participates
to the rapid and accurate diagnosis of many cancers. It relies on
the administration of an imaging probe capable of binding
specifically to a biomarker of the disease. The localization of this
probe is then classically achieved by positron emission
tomography (PET) or single photon emission computed
tomography (SPECT) in oncology.1
An interesting target involved in the development and
progression of numerous cancers is the neurotensin receptor 1
(NTS1).2,3 This G-protein coupled receptor is overexpressed in
different cancers such as pancreatic ductal adenocarcinoma,4
prostate cancer,5 Ewing’s sarcoma,6 colorectal cancer,7 breast
cancer,8 and small cell lung cancer, but not present in healthy
tissues.9 The endogenous ligand of this receptor is a
tridecapeptide called neurotensin (NT).10
These last years, neurotensin and its receptors have been the
subject of numerous studies. Most of them deal with the design
of NTS1 agonists. Those agonists are generally peptide analogs
of neurotensin, which have been labeled with radiometals or
fluorophores for nuclear imaging, radiotherapy, or fluorescence
imaging.11−14 Despite their good affinity for NTS1 (in the
nanomolar range) and interesting biodistribution properties,
these compounds often present limited metabolic stability, even
after chemical optimization.15,16
In 2013, Lang et al. synthesized an analog of SR142948A and
coupled it to 2-deoxy-2-[18F]fluoroglucosylazide to create a PET
Received: March 22, 2021
Published: June 9, 2021
In the early 90s, Sanofi-Recherche discovered a nonpeptidic
antagonist of NTS1 named SR45398 (Figure 1).17,18 After a few
https://doi.org/10.1021/acs.jmedchem.1c00523
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compound differs from 3BP-227 in its amine arm. Indeed, 3BP-
227 carries a linear amine arm, whereas 3BP-228 has a branched
amine arm (Figure 1). Tumor uptakes of [111In]In-3BP-227 and
[
111In]In-3BP-228 were similar at 3, 6, and 12 h post-injection,
but tumor-to-normal tissue ratios were generally higher for
[
111In]In-3BP-228 at the earliest time-point (3 h p.i.), although
not statistically different. Since our objective was to design a
PET tracer, showing rapid clearance from healthy tissues, in
particular from the liver and gastrointestinal tract to facilitate the
detection of the primary tumor and potential metastases, we felt
that it was more interesting to select 3BP-228’s scaffold.
Nonetheless, the compound 3BP-227 was also synthesized and
used as a reference in this study.
Gallium-68 was chosen as the radiometal. Indeed, this
positron-emitting radioisotope has a short half-life of 67.7
min, which is perfectly suited to the rapid pharmacokinetics of
small molecules and reduces patient exposure to ionizing
radiation compared to other radiometals with longer half-lives
such as copper-64 or zirconium-89. In addition, 68Ga has a
relatively low cost and is readily available thanks to 68Ge/68Ga
generators.30 Alternatively, it can be produced in cyclotrons via
the 68Zn(p,n)68Ga reaction, which further increases the
availability of this radiometal.31
Several chelating agents suitable for 68Ga complexation are
available.32−34 Among them, we first chose to evaluate NOTA,
(R)-NODAGA, DOTA, DOTAGA, and THP. All these
chelators are able to form stable complexes. Due to their
differences in charge and logD, we hypothesized that the
presence of these chelates could significantly modify the
pharmacokinetics of the tracers.35−40 In addition, we have also
chosen to introduce two (R)-NODAGA or DOTAGA chelators
on the same molecule in order to increase clearance from blood
and improve tumor-to-background ratios. This approach was
inspired by the work of Roxin et al., who showed that the
introduction of a metal-free DOTA on a 18F radiotracer could
improve tumor uptake and increase renal excretion.41
The compounds were prepared by multi-step synthesis from
four synthons (Scheme 1). The first synthon (A), 3-isopropyl-4-
hydrazinobenzoic acid hydrochloride, was synthesized in six
steps starting from 2-isopropylaniline with a yield of 9%.42 The
second synthon (B), ethyl-4-(2,6-dimethoxyphenyl)-4-hydroxy-
2-oxobut-3-enoate was prepared, starting from diethyl oxalate
and 2,6-dimethoxyacetophenone, with a yield of 73%.43 The
amine arm (synthon C) was obtained in three steps with a yield
of 50%.44 The fourth synthon (D), tert-butyl-2-aminoadaman-
tane-2-carboxylate, was synthesized in four steps starting from 2-
adamantanone with a yield of 23%.45,46 The linear amine arm
(synthon E), for the reference compound 3BP-227, was
prepared in one step in 80% yield. Four steps were then
required to obtain the precursor 16, ready for conjugation with
the different chelating agents.42,47 Finally, the introduction of
the chelators was achieved in one or two steps, in yields ranging
from 15 to 65%.
Figure 1. Chemical structures of SR45398, SR142948A, 3BP-227, and
3BP-228. DOTA stands for 1,4,7,10-tetraazacyclododecane-1,4,7,10-
tetraacetic acid.
imaging tracer.21 This compound showed an excellent affinity
for NTS1 (0.98 nM). However, despite a fairly good uptake in
tumor tissues, a significant accumulation was observed in other
organs, particularly the liver. In 2016, Schulz et al. introduced a
polyazamacrocyclic metal-chelating agent (DOTA) on different
analogs of SR142948A and radiolabeled them with indium-111,
a radionuclide suitable for SPECT imaging.26 Their compounds
[
111In]In-3BP-227 and [111In]In-3BP-228 (Figure 1) showed
good affinity for neurotensin receptor 1 and high and specific
tumor uptake in vivo. Because of its prolonged retention in the
target tissue at late time-points (24 h after injection), 3BP-227
was considered as a good candidate for the radiotherapy of
cancer. In 2017, Schulz et al., evaluated 3BP-227 (now
IPN01087) for the 177Lu-radiotherapy of colon carcinoma in a
preclinical study. Their promising results paved the way for a
radiotherapy trial,22 which was initiated in 2018 by Baum and
coll. on patients with metastatic or locally advanced cancers
expressing NTS1.27 Preliminary results provided clinical
evidence of the feasibility of treatment of ductal pancreatic
adenocarcinoma with the 177Lu-labeled NTS1 antagonist 3BP-
227.28
The availability of a PET companion diagnostic agent would
greatly facilitate the selection of patients eligible for NTS1-
targeted radiotherapy.29 In this study, we sought to assess the
influence of the chelator on the biodistribution of neurotensin
receptor antagonists. A variety of chelating agents, suitable for
the complexation of 68Ga, was coupled to an analog of
SR142948A. The resulting tracers were then evaluated in vitro
and tested in vivo on mice xenografted with an HT-29 model of
colorectal cancer or an AsPC-1 model of pancreatic cancer.
Stability. In order to evaluate the stability of these molecules
against enzymatic degradation, each compound was metalated
with non-radioactive gallium and incubated in mouse serum at
37 °C. RP-HPLC-MS analyses were performed at different time-
points (0 min, 30 min, 1 h, 2 h, 4 h, and 24 h). Interestingly,
NOTA/NODAGA/THP-based compounds proved to be stable
in these assay conditions (>99% intact product after 4 h)
whereas DOTA/DOTAGA-based compounds showed some
degradation (ca. 80% intact product after 4 h) (Table 1 and
Table S1). The main degradation corresponded to the loss of
RESULTS AND DISCUSSION
■
Design and Synthesis. A series of seven imaging agents,
antagonists of neurotensin receptors, were synthesized. In light
of the study by Schulz et al., we chose to build our compounds
based on the structure of the compound 3BP-228.26 This
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a
Scheme 1. Synthesis of NTS1 Antagonists
a
Reagents and conditions: (a) Ac2O, toluene, 0 °C to RT, 1 h; (b) NBS, DMF, RT, o.n.; (c) CuCN, H2O/DMF, reflux, o.n.; (d) 1 M HCl, EtOH,
reflux, 3 d; (e) KOH, 1,2-dimethoxyethane, reflux, 3 d; (f) (1) NaNO2, HCl/AcOH, 0 °C, 3 h, (2) SnCl2, HCl, RT, 2 h; (g) diethyl oxalate, NaH,
DMF, RT, o.n.; (h) Synthon B, AcOH, reflux, 4 d; (i) 2-nitrobenzenesulfonyl chloride, NEt3, CH2Cl2, 0 °C to RT, 15 min; (j) 3-dimethylamino-1-
propyl hydrochloride, K2CO3, DMF, 60 °C, o.n.; (k) PhSH, Cs2CO3, DMF, 40 °C, 5 h; (l) Synthon C, HATU, DIPEA, DMF, RT, 6 h; (m) NaCN,
NH4OH, NH4Cl, H2O/EtOH, 50 °C, o.n.; (n) BzCl, K2CO3, H2O/THF, RT, 2 h; (o) HCl/AcOH, reflux, 5 d; (p) t-butyl acetate, HClO4, RT,
o.n.; (q) KOH 5 M, dioxane, RT, 18 h.; (r) Synthon D, HATU, NEt3, DMF, RT, 4 h; (s) TFA/DCM 50/50, RT, 1.5 h; (t) chelating agent,
DIPEA, DMF, RT, 4 h. The values noted in parentheses are the estimated charge when each chelator is complexed to Ga3+ at pH 7.4.
gallium as evidenced by mass spectrometry. This shows the
importance of the cavity size on the thermodynamic stability of
68Ga complexes (the expansion of the macrocyclic ring results in
a decrease in the complexation constant: logKGa‑NOTA = 31.0 vs
logKGa‑DOTA = 26.1).48 After 24 h, some unidentified
degradation products (mass loss of 27) were also observed.
Although these in vitro data do not necessarily reflect the
metabolic stability of the compounds in vivo, they do confirm the
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was determined in a competition experiment on CHO cells
overexpressing the NTS1 receptor. [125I]I-Tyr3-neurotensin
(0.05 nM; Kd = 0.22 nM) was used as a ligand.49 The Ki values
were in the nanomolar range for most of the compounds (Table
1 and Figure S48). The compounds with a single chelator
showed the best affinities with Ki values between 0.7 and 3.6 nM,
similar to the reference natGa-3BP-227 (0.55 nM). According to
these results, it seems that the presence of a positive charge
favors binding to NTS1 (natGa-NOTA-16, Ki 0.73 nM), and
conversely, an increase in the number of negative charges has a
detrimental effect on affinity (natGa-DOTAGA-16, Ki 3.59 nM).
In comparison, the compounds with two chelating agents
showed a significant decrease in affinity (Ki of 9 nM and 62 nM
for natGa-bisNODAGA-16 and natGa-bisDOTAGA-16, respec-
tively).
Radiolabeling and Partition Coefficients. All the
compounds were radiolabeled with 68Ga, using [68Ga]GaCl3
obtained after elution of a generator with a 0.1 M HCl solution.
A solution of sodium acetate buffer (0.5 M, pH 5.56) was added
to the compounds in order to reach a final pH of 3.5. Ten
percent of ethanol was also introduced to limit radiolysis. The
radiolabeling of NOTA, NODAGA, and THP-based com-
pounds was performed at 37 °C for 5 min, whereas 10 min at 95
°C were required to radiolabel DOTA and DOTAGA-based
compounds. Each tracer was purified on a C18 Sep-Pack column
to obtain the desired compound with a radiochemical purity of
>94%, as determined by radio-HPLC. The radiochemical yields
ranged from 35% to 80% and molar activities from 8.8 to 17.8
MBq/nmol at the end of radiolabeling. Partition coefficients
(logD) were determined by the shake-flask method in octanol
and PBS at pH 7.4.50 All compounds were found to be
hydrophilic. As expected, compounds with two chelating agents
are more hydrophilic with logD values of about −3.5 while
values ranging from −1.7 to −2.6 are obtained for compounds
with only one chelating agent. The compound [68Ga]Ga-THP-
16 is the least hydrophilic of all with a logD of −1.2 (Table 1).
Table 1. Percentage of Inhibition Constants, Intact
Compound after 4 h, Partition Coefficients, and
Radiochemical Purities of Compounds Tested in This Study
Radiochemical
purity (radio-
HPLC)
% intact
a
b
c
compound
Ki (nM)
after 4 h
LogD
[
[
[
[
[
[
nat/68Ga]Ga-
3BP-228
1.35
80%
−2.45
−1.75
−1.85
−1.74
−1.17
−3.62
>99%
>99%
94%
[0.99−1.83]
nat/68Ga]Ga-
3.59 [3.03−
78%
DOTAGA-16
4.26]
nat/68Ga]Ga-
0.73 [0.60−
> 99%
> 99%
90%
NOTA-16
0.88]
nat/68Ga]Ga-
1.75 [0.77−
97%
NODAGA-16
3.96]
nat/68Ga]Ga-
1.80 [1.55−
99%
THP-16
2.10]
nat/68Ga]Ga-
bisDOTAGA-
16
61.9 [51.9−
78%
>99%
73.8]
[
nat/68Ga]Ga-
bisNODAGA
-16
9.05 [7.28−
> 99%
−3.44
>99%
11.2]
[
nat/68Ga]Ga-
3BP-227
0.55 [0.51−
90%
−2.62
−2.50
98%
0.59]
[
nat/68Ga]Ga-
DOTA-NT-
20.3
10.0 [6.51−
N.D.
>99%
15.4]
a
Ki values are presented as “mean [95% confidence interval]” and
were determined using natGa compound and [125I]I-Tyr3-neurotensin
b
as the radioligand. Percentage of intact compound after 4 h
incubation in mouse serum at 37 °C using natGa compounds.
c
Partition coefficients (logD) were determined at pH 7.4 using 68Ga
compounds and are presented as mean. N.D.: not determined.
good stability of small molecules relative to peptides. For
comparison, only 55% of the natural neurotensin was found
intact after 4 h of incubation in the same assay conditions (Table
S1).
In Vitro Binding Affinity. The affinity for NTS1 of the seven
natGa-metalated compounds and the reference natGa-3BP-227
a
Table 2. Ex Vivo Biodistribution and Tumor-to-Organ Ratios of 3BP-227, 3BP-228, and DOTA-NT-20.3
[68Ga]Ga-3BP-228
[68Ga]Ga-3BP-227
[68Ga]Ga-DOTA-NT-20.3
uptake (%ID/g)
n = 4
n = 4
n = 4
blood
liver
gallbladder
kidneys
spleen
heart
lungs
muscle
intestine
carcass
tumor
1.083 0.222
0.908 0.129
3.347 3.208
2.525 0.641
0.408 0.085
0.555 0.102
1.060 0.164
0.348 0.345
1.093 0.098
1.125 0.560
7.825 2.507
9.155 0.705***
3.725 2.181*
4.240 1.786
0.088 0.017*
0.103 0.022
0.108 0.071
5.705 1.457**
0.113 0.026**
0.053 0.019
0.173 0.036**
0.025 0.010
0.405 0.072*
0.160 0.022*
1.655 0.497**
4.775 1.055*
1.610 0.147***
3.518 0.843***
4.675 0.429***
0.985 0.114**
2.018 0.497**
4.253 0.621***
11.238 1.704*
tumor-to-organ
ratios
n = 4
n = 4
n = 4
blood
intestine
kidneys
7.540 2.938
1.093 0.098
3.125 0.695
1.243 0.279
2.018 0.497**
2.393 0.346
19.08 5.778**
0.405 0.072*
0.300 0.110**
a
Top: Ex vivo biodistribution of [68Ga]Ga-3BP-228, [68Ga]Ga-3BP-227, and [68Ga]Ga-DOTA-NT-20.3, 2 h p.i. Values are expressed as the
percentage of injected dose per gram of tissue (%ID/g
SD). Bottom: Tumor-to-organ ratios from the ex vivo biodistribution. Values are
expressed as means SD. Statistical analysis by one-way ANOVA with [68Ga]Ga-3BP-228 as a control group, followed by Bonferroni correction,
*p < 0.05, **p < 0.01, and ***p < 0.001.
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Figure 2. Maximum intensity projection (MIP) images obtained between 1.5 h and 2 h p.i. of [68Ga]Ga-3BP-228, [68Ga]Ga-DOTAGA-16, [68Ga]Ga-
NOTA-16, [68Ga]Ga-NODAGA-16, [68Ga]Ga-THP-16, [68Ga]Ga-bisDOTAGA-16, and [68Ga]Ga-bisNODAGA-16. The tumor is showed by a
white arrow. Abbreviations: G, gallbladder; I, intestines; K, kidneys; B, bladder; Li, liver; Lu, lungs.
Figure 3. Ex vivo biodistribution data of 68Ga compounds 2 h p.i. Values are expressed as the percentage of injected dose per gram (%ID/g, mean
SD) for each collected organ. Data for [68Ga]Ga-THP-16 are presented in Table S2.
PET-MRI Imaging and Biodistribution. In order to
evaluate the biodistribution of each compound, the radiolabeled
tracers were injected intravenously (500 pmol, 3−8 MBq) in the
lateral tail vein of nude mice bearing a xenograft of human
colorectal cancer (HT-29) in the flank. This tumor model
expresses NTS1.49 Static PET-MRI images were recorded
between 1.5 h and 2 h post-injection.
In comparison, [68Ga]Ga-DOTA-NT-20.3 showed a typical
biodistribution profile for a NTS1 peptide agonist, with a lower
uptake in the tumor compared to [68Ga]Ga-3BP-228, but a
minimal activity in all the other tissues (apart from kidneys),
giving tumor-to-organ ratios around two times higher than
[68Ga]Ga-3BP-228 for the blood, liver, muscle, and heart (Table
2 and Figure S49).
First, we chose to compare [68Ga]Ga-3BP-228 to two
references: [68Ga]Ga-3BP-227 and a well-validated NTS1
peptide agonist named [68Ga]Ga-DOTA-NT-20.3.51,52,11 As
could be expected from the results of Schulz et al. with 111In-
labeled compounds, [68Ga]Ga-3BP-228 showed faster blood
clearance than its isomer. Indeed, a high level of circulating
activity was observed for [68Ga]Ga-3BP-227, leading to a tumor-
to-blood ratio of 1.243 0.279 while a ratio of 7.540 2.938
was obtained with [68Ga]Ga-3BP-228 (Table 2 and Figure S49).
Overall, these results showed that NTS1 small molecule
antagonists have a good potential for 68Ga-PET diagnosis but
that their pharmacokinetics needed to be further optimized.
Next, we undertook the evaluation of our seven novel
derivatives. All these compounds showed important differences
in terms of biodistribution. For instance, administration of the
THP derivative gave a much stronger signal in the lungs, spleen,
and liver (12.7 4.1%ID/g, 46.4 9.3%ID/g, and 42.0 8.1%
ID/g, respectively) than all the other compounds (0.5−1.3%ID/
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a
Table 3. Ex Vivo Biodistribution and Tumor-to-Organ Ratios at 2 h p.i.
[68Ga]Ga-3BP
-228
[68Ga]Ga-DOTAGA
[68Ga]Ga-
NODAGA-16
[68Ga]Ga-
bisDOTAGA-16
[68Ga]Ga-
bisNODAGA-16
[68Ga]Ga-DOTA-
NT-20.3
-16
[68Ga]Ga-NOTA-16
uptake
(%ID/g)
n = 4
n = 4
n = 4
n = 4
n = 4
n = 4
n = 4
blood
1.083 0.222
0.908 0.129
3.347 3.208
2.525 0.641
0.408 0.085
0.555 0.102
1.060 0.164
0.348 0.345
1.093 0.098
1.125 0.560
7.825 2.507
0.658 0.039
0.523 0.025
2.803 1.062
1.943 0.525
0.310 0.174
0.335 0.019
0.678 0.194
0.205 0.044
0.678 0.127
1.043 0.474
3.978 0.564***
1.330 0.654
1.183 0.325
-
1.915 0.526
1.005 0.211
10.610 12.404
3.775 1.811
0.448 0.141
0.750 0.249
1.285 0.452
1.475 0.997**
4.293 0.483
1.603 0.496
8.148 1.038
0.135 0.031*
0.250 0.057
0.367 0.040
3.353 0,523
0.118 0.019
0.103 0.021**
0.255 0.031
0.138 0.083
0.183 0.083
0.313 0.036
1.305 0.062***
0.393 0.108
0.387 0.072
1.363 0.993
3.377 0.425
0.207 0.025
0.167 0.038*
0.437 0.071
0.210 0.061
0.267 0.097
0.610 0.154
4.917 0.776*
0.088 0.017*
0.103 0.022
0.108 0.071
5.705 1.457**
0.113 0.026**
0.053 0.019
0.173 0.036**
0.025 0.010
0.405 0.072*
0.160 0.022*
1.655 0.497**
liver
gallbladder
kidneys
spleen
2.540 0.713
0.445 0.177
0.518 0.205
1.165 0.527
0.413 0.114
4.080 1.272
1.225 0.513
3.140 0.286***
heart
lungs
muscle
intestine
carcass
tumor
tumor-to-organ
ratios
n = 4
n = 4
n = 4
n = 4
n = 4
n = 4
n = 4
blood
7.540 2.938
6.093 1.173
6.125 2.026
2.158 0.655
2.788 1.172*
0.808 0.167*
1.288 0.244**
4.468 1.085
1.900 0.177
2.555 1.116
10.028 2.189
8.843 5.184
0.393 0.051
13.190 4.216*
19.747 5.633***
1.450 0.053**
19.083 5.778**
4.290 1.899
intestine
kidneys
7.145 2.064
3.125 0.695
0.300 0.110***
a
Values are expressed as mean SD. Data for THP-16 are shown in Tables S2 and S3. Statistical analysis by one-way ANOVA with [68Ga]Ga-3BP-
228 as a control group, followed by Bonferroni correction, *p < 0.05, **p < 0.01, and ***p < 0.001.
g) (Figure 2). Moreover, the tumor uptake of [68Ga]Ga-THP-
16 was very low (1.33 0.35%ID/g) (Tables S2 and S3 and
Figure S49). We assume that this behavior results from the
lipophilicity of the THP moiety.53 Indeed, [68Ga]Ga-THP-16
has a logD of −1.17 while the other compounds have logD
values between −1.75 and −3.62 (Table 1).
(Figures 2 and 3 and Table 3). Interestingly, this compound did
not accumulate in the gallbladder and intestines.
In order to evaluate the specificity for NTS1, a blocking
experiment (in the presence of a 100-fold molar excess of
SR142948A) was carried out with the tracers [68Ga]Ga-
bisNODAGA-16, [68Ga]Ga-DOTAGA-16, and [68Ga]Ga-
3BP-228. The uptake in all isolated organs remained essentially
unchanged, apart from tumor uptake whose value decreased by a
factor of 2 to 5 (Figure 4 and Table S4). PET-MRI imaging
confirmed the biodistribution results (Figure S50). These
results demonstrate the specificity of these tracers for neuro-
tensin receptor-positive tumors.
The other four compounds, labeled with a single macrocyclic
chelator, gave fairly similar profiles for most organs. The main
difference was observed in the gallbladder and intestines
(Figures 2 and 3). Indeed, the NOTA/NODAGA compounds
showed a much stronger signal in these organs than DOTA/
DOTAGA compounds, suggesting a partial hepatobiliary
excretion pathway for these derivatives. This may limit their
interest for the imaging of abdominal tumors. Concerning tumor
uptake, we observed on PET images that the compounds
[68Ga]Ga-NODAGA-16 and [68Ga]Ga-3BP-228 showed a
better accumulation in the tumor than [68Ga]Ga-NOTA-16
and [68Ga]Ga-DOTAGA-16 (Figure 2). Indeed, for the
NODAGA- and DOTA-based compound, a signal of about
8%ID/g 2 h p.i. was observed while the NOTA- and DOTAGA-
based compounds gave only 3−4%ID/g (Figure 3). However,
the best tumor-to-organ ratios were observed for the tracers
[68Ga]Ga-3BP-228 and [68Ga]Ga-DOTAGA-16 (Table 3).
Next, the introduction of two chelating agents on the same
molecule was attempted with the objective to speed up the
clearance of the tracer from non-targeted tissues. The
corresponding PET images showed high contrast, with less
than 0.4%ID/g in blood after 2 h (Figure 2). The bisDOTAGA
derivative, [68Ga]Ga-bisDOTAGA-16, showed very little signal
in all organs but also a rather low tumor uptake (1.305 0.062%
ID/g), leading to tumor-to-organ ratios quite similar to those
obtained with the corresponding single-chelator [68Ga]Ga-
DOTAGA-16 (Figure 3 and Table 3). We assume that the
limited retention in the tumor tissue is probably related to its
reduced affinity for NTS1 (Ki = 61.9 nM) compared to the other
molecules. In contrast, [68Ga]Ga-bisNODAGA-16 showed an
attractive biodistribution profile with less signal in all non-
targeted organs while retaining a good tumor uptake value of
4.917 0.776%ID/g, leading to the best tumor-to-organ ratios
In Vitro and In Vivo Evaluation of [68Ga]Ga-bisNODA-
GA-16. In order to determine whether the uptake of the tracer
bisNODAGA-16 in HT29 tumor was mediated by NTS1, NTS2,
Figure 4. Ex vivo biodistribution of [68Ga]Ga-bisNODAGA-16 2 h p.i.,
with or without a 100-fold excess of SR142948A. Data were analyzed by
an unpaired two-tailed Student’s t-test (***p < 0.001).
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or both receptors, an in vitro competition assay on HT29 cells
was performed (Figure S51). The binding of [68Ga]Ga-
bisNODAGA-16 to HT29 cells was inhibited by an excess of
SR142948A or neurotensin, but not by levocabastine, a
histamine H1 receptor antagonist that blocks NTS2.20 These
results suggest that the tumor uptake observed in vivo, in this
model, is primarily due to binding to NTS1. The antagonistic
properties of the lead compound natGa-bisNODAGA-16 were
confirmed in a calcium flux mobilization assay performed on
CHO cells expressing human recombinant NTS1 (IC50 = 141
nM; KB = 13 nM; Figure S52).
lipophilicity (or lack of in vivo stability), which leads to
unfavorable hepatobiliary clearance. Several studies have
suggested that the charge of the chelate would have a major
impact on the biodistribution.39,58,36 In our specific case,
compounds with neutral charge complexes presented a better
tumor accumulation in vivo.
On a positive note, it appears that the addition of an extra-
chelator is a reliable and straightforward strategy to speed up the
renal excretion of tracers, leading to better images, provided that
the affinity is retained.41 In addition, it increases the molar
activities that can be potentially achieved. However, it should be
noted that this approach may complicate the pharmaceutical
development of the tracer as it is difficult to predict which
chelator binds to the radiometal. Saturation of the molecule with
natGa, after radiolabeling, may be necessary to obtain a
homogeneous tracer.
[68Ga]Ga-bisNODAGA-16 was further evaluated in a model
of pancreatic ductal adenocarcinoma (subcutaneous xenografts
of human AsPC-1 tumor cells).11 PET/CT images were
recorded 50−70 min and 90−110 min after injection (Figure
S53). Tumor masses could be easily detected as early as 60 min
after injection. A post-mortem biodistribution study was
performed 2 h post-injection, which confirmed the accumu-
lation of the radioactive tracer in the tumor (Table S5).
EXPERIMENTAL SECTION
■
General Information. All chemicals were purchased from Merck,
Acros Organics, and Alfa Aesar and used without further purification.
DOTA-NHS, DOTAGA-anhydride, NOTA-NHS, (R)-NODAGA-
NHS, THP-NCS, DOTAGA(tBu)4, and NODAGA(tBu)3 were
provided by CheMatech (Dijon, France). Moisture-sensitive reactions
were performed under a nitrogen or argon atmosphere. The purity of
final compounds was determined by RP-HPLC-MS and was >95% for
all tested compounds unless stated.
CONCLUSIONS
■
Since the discovery of NTS1 receptor overexpression in many
tumors, considerable efforts have been undertaken to develop
NTS1-targeted radiopharmaceuticals that may improve cancer
diagnosis and stratification (the reader is encouraged to refer to
Maschauer and Prante’s excellent review on the topic).12
Unfortunately, despite extensive optimization work, a very few
tracers based on neurotensin peptide analogs have been tested in
patients, and their results have so far been disappointing.16,54,55
In this context, the emergence of nonpeptide antagonist
radioligands of NTS1 is a new source of hope. While efforts
have until now been concentrated on 177Lu-labeling of these
compounds for internal vectorized radiotherapy, here we
investigated whether they could be adapted to the elaboration
of NTS1-specific 68Ga-PET imaging tracers to form a theranostic
pair.
Experimental Procedures and Characterization Data for
Synthon A. 2-Isopropylacetanilide (1). A solution containing 2-
isopropylaniline (5 mL, 35.3 mmol, 1 equiv) and toluene (48 mL) was
cooled in an ice bath. Acetic anhydride (3.52 mL, 37.1 mmol, 1.05
equiv) was added slowly. After stirring for 45 min at RT, the reaction
medium was evaporated. The red oil obtained was then taken up with
petroleum ether (15 mL). The white precipitate formed was filtered and
dried under vacuum to give a white solid (5.83 g, 93%, purity >99%).
RP-HPLC-MS: tr = 3.82 min, m/z calculated for C11H15NO [M + H]+
178.1, found 178.0. Rf (CH2Cl2/MeOH, 98/2) = 0.26. 1H-NMR (500
MHz, CDCl3): δ = 1.25 (d, J = 6.8 Hz, 6H), 2.2 (s, 3H), 3.05 (hept, J =
6.8 Hz, 1H), 7−7.3 (m, 4H), 7.6 (m, 1H) ppm; 13C-NMR (126 MHz,
CDCl3): δ = 23.2 (CH3), 24.3 (CH3), 28.1 (CH), 125.4 (CH), 125.8
(CH), 126.4 (CH), 126.5 (CH), 134.1 (C), 141.1 (C), 168.9 (C) ppm.
mp: 72 °C.
4-Bromo-2-isopropylacetanilide (2). A solution of N-bromosucci-
nimide (7.53 g, 42.3 mmol, 1.5 equiv) in DMF (14 mL) was added
dropwise over 45 min to a solution of 2-isopropylacetanilide (1) (5 g,
28.2 mmol, 1 equiv) in DMF (14 mL). The mixture was stirred at RT
overnight. The reaction mixture was evaporated to obtain a yellow oil,
extracted with CH2Cl2, washed with acidic water and brine, and dried
over MgSO4. The solvent was evaporated to give a yellow solid, which
was purified by flash-column chromatography (A: CH2Cl2, B: CH2Cl2/
MeOH 9:1; with the following gradient program: 0% to 40% of B in 25
CV). A white solid (4.62 g, 64%, purity: 93%) was obtained. RP-HPLC-
MS: tr = 4.26 min, m/z calculated for C11H14BrNO [M − H]− 254.0,
found 254.0. Rf (CH2Cl2/MeOH, 98/2) = 0.32. 1H-NMR (500 MHz,
CDCl3): δ = 1.23 (d, J = 6.8 Hz, 6H), 2.19 (s, 3H), 2.98 (hept, J = 6.8
Hz, 1H), 7.00 (br, 1H), 7.30 (d, J = 8.5 Hz, 1H), 7.38 (s, 1H), 7.53 (d, J
= 8.5 Hz,1H) ppm; 13C-NMR (126 MHz, CDCl3): δ = 23.0 (CH3),
24.3 (CH3), 28.2 (CH), 119.7 (C), 126.8 (CH), 129.0 (CH), 129.6
(CH), 133.2 (C), 143.0 (C), 168.8 (C) ppm. mp: 132 °C.
In a seminal paper published in 2012, Fani et al. stated that
“the chelate makes the difference”, stressing that the structure of
a chelating agent can greatly influence the biodistribution of a
tracer.56 Since then, numerous teams confirmed this observa-
tion.35−38,40,57−61 By evaluating seven different chelating agents
introduced on the same NTS1 antagonist molecule, we were able
to identify a promising new PET tracer, [68Ga]Ga-bisNODA-
GA-16, that showed rapid accumulation in the tumor (4.917
0.776%ID/g, 2 h p.i.) and fast clearance from blood and other
organs. This behavior led to contrasted PET-MRI images, with
tumor-to-organ ratios of 1.45 0.05, 13.2 4.2, to 24.9 8.6
for the kidneys, blood, and muscle, respectively. This antagonist
showed a biodistribution profile similar to that of the agonist
peptide analogue [68Ga]Ga-DOTA-NT-20.3, with comparable
or even better tumor-to-organ ratios for organs such as kidneys,
spleen, heart, lungs, and intestines.
Unfortunately, to date, the effect of the chelating agent on the
pharmacokinetics of a tracer is still hard to predict and needs to
be evaluated in vivo. For example, although a study by Young’s et
al. has found that the introduction of THP on a PSMA
radiotracer could give very good results,62 in our study, the THP
derivative showed poor tumor uptake and high accumulation in
the spleen, kidneys, and lungs. While tracers such as [68Ga]Ga-
JMV6659 show NTS1-mediated accumulation in the spleen and
blood (which the authors suggest is related to specific binding to
NTS1 expressed on blood cells),63 we believe that the observed
biodistribution for [68Ga]Ga-THP-16 results primarily from its
4-Cyano-2-isopropylacetanilide (3). A mixture of 4-bromo-2-
isopropylacetanilide (2) (4.8 g, 18.74 mmol, 1 equiv), DMF (22.5
mL), cuprous cyanide (2.51 g, 28.1 mmol, 1.5 equiv), and water (0.370
mL) was stirred under reflux overnight. After cooling, the reaction
medium was concentrated under vacuum and water (58 mL) was added
to the mixture under stirring. The precipitate was filtered off, rinsed
with water, and dried under vacuum. The gray/brown solid was purified
by flash-column chromatography (A: CH2Cl2, B: CH2Cl2/MeOH 9:1;
with the following gradient program: 0% to 40% of B in 25 CV). A white
solid (1.23 g, 33%, purity: 95%) was obtained. RP-HPLC-MS: tr = 3.65
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min, m/z calculated for C12H14N2O [M − H]− 201.1, found 201.1. 1H-
NMR (500 MHz, CDCl3): δ = 1.28 (d, J = 6.8 Hz, 6H), 2.24 (s, 3H),
3.01 (hept, J = 6.8 Hz, 1H), 7.25 (br, 1H), 7.49 (d, J = 8.5 Hz, 1H), 7.54
(s, 1H), 8.08 (d, J = 8.5 Hz, 1H) ppm; 13C-NMR (126 MHz, CDCl3): δ
= 22.6 (CH3), 24.7 (CH3), 27.8 (CH), 108.4 (C), 119.1 (C), 123.4
(CH), 129.8 (CH), 130.5 (CH), 138.5 (C), 168.4 (C) ppm. mp: 139
°C.
4-Cyano-2-isopropylaniline Hydrochloride (4). A mixture of 4-
cyano-2-isopropylacetanilide (3) (2.5 g, 15.3 mmol, 1 equiv), absolute
ethanol (12 mL), and 1 N HCl (12 mL) was stirred under reflux for 3
days. A solution of 10% NaOH (7 mL) was added to reach a pH of 10.
The reaction medium was extracted three times with CH2Cl2, and the
organic layers were combined, dried over MgSO4, and concentrated
under vacuum. The orange oil obtained was dissolved in diethyl ether
(36 mL), and 1 M ethereal hydrogen chloride (10 mL) was added. The
white precipitate was filtered off and rinsed with diethyl ether to give the
expected product (2.21 g, 74%, purity: 91%). RP-HPLC-MS: tr = 4.22
min, m/z calculated for C10H13N2+ [M + CH3CN]+ 202.3, found 202.1.
1H-NMR (500 MHz, DMSO-d6): δ = 1.14 (d, J = 6.8 Hz, 6H), 2.96
(hept, J = 6.8 Hz, 1H), 6.72 (d, J = 8.3 Hz, 1H), 7.28 (d, J = 8.3 Hz, 1H),
7.34 (s, 1H) ppm; 13C-NMR (126 MHz, DMSO-d6): δ = 22.4 (CH3),
26.7 (C), 107.4 (C), 115.1 (C), 121.3 (CH), 129.7 (CH), 131.1 (CH)
ppm. mp: 186 °C.
4-Amino-3-isopropylbenzoic Acid Hydrochloride (5). A solution of
potassium hydroxide (16.8 g, 0.3 mol, 20 equiv), water (18 mL), and
1,2-dimethoxyethane (1.5 mL) was added to 4-cyano-2-isopropylani-
line hydrochloride (4) (3.0 g, 15.0 mmol, 1 equiv). The mixture was
heated to reflux for 3 days. After cooling, concentrated HCl (12 mL)
was added to reach a pH of 1 and the mixture was then extracted three
times with CH2Cl2. The organic layers were combined, dried over
MgSO4 and concentrated to yield an orange solid (2.42 g, 75%, purity:
97%). RP-HPLC-MS: tr = 3.33 min, m/z calculated for C10H13NO2 [M
+ H + CH3CN]+ 221.1, found 221.0. Rf (CH2Cl2/MeOH, 98/2) = 0.3.
1H-NMR (500 MHz, CDCl3): δ = 1.30 (d, J = 6.8 Hz, 6H), 2.85 (hept, J
3.82 (s, 6H), 4.35 (q, J = 7.1 Hz, 2H), 6.59 (d, J = 8.4 Hz, 2H), 6.61 (s,
1H), 7.34 (t, J = 8.4 Hz, 1H), 14.33 (br 1H) ppm; 13C-NMR (126
MHz, CDCl3): δ = 14.2 (CH3), 56.2 (CH3), 62.5 (CH2), 104.2 (CH),
106.4 (CH), 117.2 (C), 132.4 (CH), 158.0 (C), 162.6 (C), 163.9 (C),
195.9 (C) ppm. mp: 103 °C.
Experimental Procedures and Characterization Data for 6. 4-
(5-(2,6-Dimethoxyphenyl)-3-(ethoxycarbonyl)-1H-pyrazol-1-yl)-3-
isopropylbenzoic Acid (6). A mixture of 3-isopropyl-4-hydrazinoben-
zoic acid hydrochloride (synthon A) (0.56 g, 2.4 mmol, 1.05 equiv) and
ethyl-4-(2,6-dimethoxyphenyl)-4-hydroxy-2-oxobut-3-enoate (syn-
thon B) (0.65 g, 2.3 mmol, 1 equiv) in AcOH (8.5 mL) was stirred
under reflux for 4 days. The brownish suspension was poured into 15
mL of ice-water bath, and the yellow precipitate was filtered off and
washed with water. The yellow solid was purified on a silica plug
(CH2Cl2 to CH2Cl2/MeOH 9:1) to obtain the expected product (0.8 g,
80%, purity: 92%). RP-HPLC-MS: tr = 5.1 min, m/z calculated for
C24H26N2O6 [M + H]+ 439.2, found 439.2. Rf (CH2Cl2/MeOH, 9/1) =
0.52. 1H-NMR (500 MHz, DMSO-d6): δ = 0.96 (d, J = 5.3 Hz, 6H),
1.31 (t, J = 7.1 Hz, 3H), 2.65 (m, 1H), 3.61 (s, 6H), 4.31 (q, J = 7.1 Hz,
2H), 6.60 (d, J = 8.5 Hz, 2H), 6.83 (s, 1H), 7.28 (d, J = 8.2 Hz, 1H),
7.31 (t, J = 8.5 Hz, 1H), 7.76 (dd, J = 8.2, 1.8 Hz, 1H), 7.85 (d, J = 1.8
Hz, 1H), 13.19 (br, 1H) ppm; 13C-NMR (126 MHz, DMSO-d6): δ =
14.7 (CH3), 27.6 (CH3), 40.6 (CH), 56.0 (CH3), 60.8 (CH2), 104.3
(CH), 106.1 (C), 111.2 (CH), 127.0 (CH), 127.4 (CH), 128.6 (CH),
132.20 (C), 132.2 (CH), 139.2 (C), 141.2 (C), 143.7 (C), 146.0 (C),
158.4 (C), 162.2 (C), 167.1 (C) ppm.
Experimental Procedures and Characterization Data for
3BP-227. Benzyl Methyl(3-(methyl(3-(methylamino)propyl)-
amino)propyl)carbamate (synthon E). A solution of N,N-bis[3-
(methylamino)propyl]methylamine (2 mL, 11 mmol, 5 equiv) in
CH2Cl2 (15 mL) was cooled to −78 °C. To this solution was added
dropwise a solution of benzyl chloroformate (0.3 mL, 2.2 mmol, 1
equiv) in CH2Cl2 (6 mL) over 1 h. The reaction was stirred at −78 °C
for 1.5 h before warming to room temperature and stirred for another
15 h. The white suspension obtained was washed twice with a solution
of K2CO3 5%, twice with brine, dried over MgSO4, filtered, and
concentrated under vacuum. The colorless oil obtained was purified by
semi-preparative RP-HPLC. A colorless oil (0.542 g, 80%, purity: 94%)
was isolated. RP-HPLC-MS: tr = 0.32 min, m/z calculated for
= 6.8 Hz, 1H), 6.66 (d, J = 8.3 Hz, 1H), 7.80 (dd, J = 8.3, 1.9 Hz, 1H),
7.92 (d, J = 1.9 Hz, 1H) ppm; 13C-NMR (126 MHz, CDCl3): δ = 22.1
(CH3), 27.8 (CH), 114.7 (CH), 119.1(C), 128.5 (CH), 129.8 (CH),
131.4 (C), 148.9 (C), 172.5 (C) ppm. mp: 130 °C.
3-Isopropyl-4-hydrazinobenzoic Acid Hydrochloride (Synthon A).
A solution of concentrated HCl (25 mL) and AcOH (22 mL) was
added to 4-amino-3-isopropylbenzoic acid hydrochloride (5) (1.3 g, 6.0
mmol, 1 equiv). The mixture was cooled to −5 °C, and a solution of
NaNO2 (0.62 g, 9.0 mmol, 1.5 equiv) in water (4.5 mL) was added
dropwise over 10 min. The mixture was stirred at 0 °C for 3 h and then
cooled to −10 °C. A solution of stannous chloride dihydrate (4.11 g,
18.0 mmol, 3.1 equiv) in concentrated HCl (4.5 mL) was added slowly.
The temperature was allowed to rise to RT, and the precipitate formed
was filtered off after 2 h and rinsed with 1 M HCl (5 mL). A yellowish
powder (1.29 g, 93%, purity: 89%) was obtained after drying over P2O5.
RP-HPLC-MS: tr = 0.4 min, m/z calculated C10H14N2O2 [2 M + H]+
389.2, found 389.2. 1H-NMR (500 MHz, DMSO-d6): δ = 1.19 (d, J =
6.8 Hz, 6H), 3.14 (hept, J = 6.8 Hz, 1H), 6.98 (d, J = 8.4 Hz, 1H), 7.76
(d, J = 8.4 Hz, 1H), 7.78 (s, 1H), 8.31 (br, 1H) ppm; 13C-NMR (126
MHz, DMSO-d6): δ = 22.9 (CH3), 26.5 (CH), 112.6 (CH), 124.0 (C),
127.0 (CH), 128.5 (CH), 134.7 (C), 146.1 (C), 167.6 (C) ppm. mp:
245 °C.
Experimental Procedures and Characterization Data for
Synthon B. Ethyl-4-(2,6-Dimethoxyphenyl)-4-hydroxy-2-oxobut-3-
enoate (Synthon B). Sodium hydride 60% (0.40 g, 9.96 mmol, 1.2
equiv) was added to a solution of diethyl oxalate (1.36 g, 9.96 mmol, 1.2
equiv) in DMF (5 mL), and the mixture was stirred for 5 min. A
solution of 2,6-dimethoxyacetophenone (1.5 g, 8.3 mmol, 1 equiv) in
DMF (5 mL) was slowly added over 15 min. The mixture was then
stirred at RT overnight. The reaction medium was extracted with
EtOAc three times, ensuring that the pH remained below 6 between
each extraction. The organic layers were combined, dried over MgSO4,
and concentrated under vacuum. Following recrystallization from
EtOH, a yellowish powder (1.7 g, 73%, purity: 90%) was obtained. RP-
HPLC-MS: tr = 4.90 min, m/z calculated for C14H16O6 [M + H]+ 281.1,
found 281.1. 1H-NMR (500 MHz, CDCl3): δ = 1.37 (t, J = 7.1 Hz, 3H),
1
C17H29N3O2 [M + H]+ 308.2, found 308.2. H-NMR (500 MHz,
CDCl3): δ = 1.95 (m, 2H), 2.21 (m, 2H), 2.66 (s, 3H), 2.76 (m, 4H),
+
2.91 (s, 3H), 3.07 (m, 5H), 3.34 (m, 2H), 5.09 (s, 2H), 5.76 (br, NH2 ),
7.3−7.4 (m, 5H), 9.30 (br, NH+) ppm. 13C-NMR (126 MHz, CDCl3):
δ = 21.1 (CH2), 22.4 (CH2), 33.3 (CH3), 39.8 (CH3), 45.9 (CH3), 46.2
(CH2), 53.1 (CH2), 54.3 (CH2), 67.6 (CH2), 116.0 (q, TFA), 127.6
(CH), 128.2 (CH), 128.6 (CH), 136.4 (C), 157.2 (C), 161.3 (q, TFA)
ppm.
Ethyl 1-(4-((3-((3-(((Benzyloxy)carbonyl)(methyl)amino)propyl)-
(methyl)amino)propyl)(methyl)carbamoyl)-2-isopropylphenyl)-5-
(2,6-dimethoxyphenyl)-1H-pyrazole-3-carboxylate (7). To a solution
of 6 (200 mg, 0.46 mmol, 1 equiv) in anhydrous DMF (2 mL) was
added DIPEA (96 μL, 0.55 mmol, 1.2 equiv) and HATU (228 mg, 0.6
mmol, 1.3 equiv). The solution was stirred 20 min at RT. A solution of
benzyl methyl(3-(methyl(3-(methylamino)propyl)amino)propyl)-
carbamate (TFA salt) (synthon E) (169 mg, 0.55 mmol, 1.2 equiv)
in DMF (2 mL), and DIPEA (289 μL, 1.66 mmol, 3.6 equiv) was added
to the reaction mixture. The yellow solution was stirred 7 h at RT. The
reaction mixture was evaporated to obtain a brown oil, which was taken
up in CH2Cl2 and washed four times with brine. The organic layer was
dried over MgSO4, filtered, and concentrated under vacuum. The oil
was purified by flash-column chromatography (A: CH2Cl2, B: MeOH;
with the following gradient program: 0% to 100% of B in 30 CV). An
orange oil was obtained (110 mg, 33%, purity: 98%). RP-HPLC-MS: tr
= 4.55 min, m/z calculated for C41H53N5O7 [M + H]+ 728.9, found
728.4. Rf (CH2Cl2/MeOH, 95/5) = 0.23. 1H-NMR (500 MHz,
CDCl3): δ = 0.96 (s, 6H), 1.39 (t, J = 7.1 Hz, 3H), 2.00 (m, 2H), 2.06
(m, 2H), 2.71 (m, 1H), 2.76 (m, 2H), 2.90 (s, 3H), 2.93 (s, 3H), 3.12
(m, 5H), 3.38 (m, 2H), 3.55 (m, 2H), 3.63 (s, 6H), 4.41 (q, J = 7.1 Hz,
2H), 5.10 (s, 2H), 6.44 (d, J = 8.3 Hz, 2H), 6.92 (s, 1H), 7.2−7.4 (m,
9H) ppm. 13C-NMR (126 MHz, CDCl3): δ = 12.5 (CH3), 14.4 (CH3),
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21.8 (CH2), 22.6 (CH2), 25.4 (CH), 27.6 (CH3), 34.3 (CH3), 37.7
(CH3), 39.4 (CH3), 43.5 (CH2), 44.4, 45.6 (CH2), 50.8 (CH3), 53.4,
53.8 (CH2), 55.5 (CH3), 60.9 (CH2), 103.5 (CH), 106.5 (C), 111.2
(CH), 116.6 (CH), 124.3 (CH), 124.9 (CH), 127.7 (CH), 128.2
(CH), 128.6 (CH), 131.6 (C), 135.8 (C), 136.5 (C), 139.1 (C), 139.4
(C), 143.9 (C), 146.7 (C), 158.3 (C), 162.8 (C), 175.0 (C) ppm.
1-(4-((3-((3-(((Benzyloxy)carbonyl)(methyl)amino)propyl)-
(methyl)amino)propyl)(methyl)carbamoyl)-2-isopropylphenyl)-5-
(2,6-dimethoxyphenyl)-1H-pyrazole-3-carboxylic Acid (8). To a
solution of 7 (0.1 g, 0.14 mmol, 1 equiv) in dioxane (2 mL) was
added a solution of 5 M KOH (0.274 mL, 1.4 mmol, 10 equiv). The
yellow solution was stirred overnight at 40 °C. The reaction mixture
was concentrated under vacuum. The residue was dissolved in water
and acidified to a pH of 2 with 1 M HCl. The aqueous phase was
extracted three times with CH2Cl2, dried over MgSO4, filtered, and
concentrated under vacuum to obtain a yellow oil (91 mg, 95%, purity:
98%). RP-HPLC-MS: tr = 4.12 min, m/z calculated for C39H49N5O7 [M
+ H]+ 700.3, found 700.3. 1H-NMR (500 MHz, CDCl3): δ = 0.96 (m,
6H), 2.03 (m, 4H), 2.69 (m, 1H), 2.77 (m, 2H), 2.90 (s, 3H), 2.93 (s,
3H), 3.0−3.2 (m, 5H), 3.4 (m, 2H), 3.5 (m, 2H), 3.6 (s, 6H), 5.11 (s,
2H), 6.44 (d, J = 8.3 Hz, 2H), 6.95 (s, 1H), 7.2−7.4 (m, 9H) ppm.
2-(5-(2,6-Dimethoxyphenyl)-1-(2-isopropyl-4-(methyl-(3-(meth-
yl-(3-(methylamino)propyl)amino)propyl)carbamoyl)phenyl)-1H-
pyrazole-3-carboxamido)adamantane-2-carboxylic Acid (9). To a
solution of 8 (75 mg, 0.11 mmol, 1 equiv) in DMF (2 mL) was added
NEt3 (45 μL, 3.21 mmol, 3 equiv) and HATU (44.9 mg, 0.12 mmol, 1.1
equiv). The yellowish solution was stirred 20 min at RT. A solution of
tert-butyl-2-aminoadamantane-2-carboxylate (synthon D) (29.7 mg,
0.12 mmol, 1.1 equiv) in DMF (1 mL) and NEt3 (29.8 μL, 0.21 mmol, 2
equiv) was added, and the reaction mixture was stirred for 4 h at RT.
The reaction mixture was concentrated under vacuum to obtain an
orange oil. The intermediate was hydrogenated in EtOH (10 mL) and
Pd/C 10% under atmospheric pressure of H2. The suspension was
stirred at RT for 2 days. The reaction mixture was filtered on celite and
concentrated under vacuum to give an oil. Finally, CH2Cl2 (1 mL), TFA
(1 mL), and H2O (0.1 mL) were added. The yellow solution was stirred
at RT for 1.5 h. The reaction mixture was concentrated under vacuum
and purified by semi-preparative RP-HPLC on a BetaBasic-18 column
(A: H2O 0.1% TFA, B: MeCN 0.1% TFA with the following gradient
program: 30% of B for 5 min, 30% to 60% of B in 50 min, at a flow rate of
20 mL/min) to obtain the product as a white powder (2 TFA salt, 29
mg, 27%, purity >99%). RP-HPLC-MS: tr = 3.75 min, m/z calculated
C58H84N10O13 [M + H]+ 1129.62987, found 1129.63250.; m/z
calculated for [M + 2H]2+ 565.30309, found 565.31998.
Experimental Procedures and Characterization Data for
Synthon C. tert-Butyl (3-((2-Nitrophenyl)sulfonamido)propyl)-
carbamate (10). A solution of N-Boc-1,3-propanediamine (0.5 g,
2.87 mmol, 1 equiv), CH2Cl2 (7 mL), and triethylamine (0.4 mL, 2.87
mmol, 1 equiv) was cooled in an ice-water bath. 2-Nitrobenzene-
sulfonyl chloride (0.636 g, 2.87 mmol, 1 equiv) was added over a period
of 5 min. After 5 min, the ice bath was removed and the reaction mixture
was allowed to warm to room temperature and stirred for 15 min. The
reaction mixture was extracted with CH2Cl2 and washed three times
with a solution of citric acid (pH = 3). The organic layer was dried over
MgSO4, filtered, and concentrated under vacuum. The yellowish oil was
purified by flash-column chromatography (A: CH2Cl2, B: CH2Cl2/
MeOH 9:1; with the following gradient program: 100% to 0% of A in 20
CV). An oil, which crystallizes, was obtained (0.942 g, 90%, purity:
98%). RP-HPLC-MS: tr = 4.46 min, m/z calculated for C14H21N3O6S
[M + H]+ 360.1, found 260.1 [M − Boc + H]+. Rf (CH2Cl2/MeOH, 9/
1) = 0.68. 1H-NMR (500 MHz, CDCl3): δ = 1.42 (s, 9H), 1.69 (quint, J
= 6.4 Hz, 2H), 3.15 (q, J = 6.4 Hz, 2H), 3.21 (q, J = 6.4 Hz, 2H), 4.67
(br, 1H), 5.87 (br, 1H), 7.72 (m, 2H), 7.84 (m, 1H), 8.16 (m, 1H)
ppm; 13C-NMR (126 MHz, CDCl3): δ = 28.4 (CH3), 30.6 (CH2), 37.2
(CH2), 40.8 (CH2), 125.3 (CH), 130.9 (CH), 132.7 (CH), 133.4
(CH) ppm. (Note: three quaternary carbons not detected).
tert-Butyl (3-((N-(3-(Dimethylamino)propyl)-2-nitrophenyl)-
sulfonamido)propyl)carbamate (11). To a solution of tert-butyl (3-
((2-nitrophenyl)sulfonamido)propyl)carbamate (10) (0.94 g, 2.6
mmol, 1 equiv) in DMF (10 mL) was added K2CO3 (1.08 g, 7.8
mmol, 3 equiv). To the stirred suspension was added 3-dimethylamino-
1-propyl hydrochloride (0.45 g, 2.86 mmol, 1.1 equiv) over a period of
5 min. The resulting suspension was heated to 60 °C overnight. The
reaction mixture was cooled to RT, diluted with water (50 mL), and
extracted three times with CH2Cl2. The organic layers were combined,
washed with brine, dried over MgSO4, filtered, and concentrated under
vacuum. A yellowish oil (1.15 g, 99%, purity: 96%) was obtained. RP-
HPLC-MS: tr = 3.6 min, m/z calculated for C19H32N4O6S [M + H]+
1
445.2, found 445.2. H-NMR (500 MHz, CDCl3): δ = 1.41 (s, 9H),
1.66 (quint, J = 6.9 Hz, 2H), 1.74 (quint, J = 6.9 Hz, 2H), 2.13 (s, 6H),
2.19 (t, J = 7.0 Hz, 2H), 3.14 (m, 2H), 3.32 (t, J = 7.4 Hz, 2H), 3.36 (t, J
= 7.4 Hz, 2H), 4.91 (br, 1H), 7.62 (m, 2H), 7.98 (m, 2H) ppm; 13C-
NMR (126 MHz, CDCl3): δ = 26.5 (CH2), 28.4 (CH3), 37.3 (CH2),
45.3 (CH2), 45.4 (CH3), 45.9 (CH2), 56.5 (CH2), 79.2 (C), 124.2
(CH), 130.6 (CH), 131.7 (CH), 133.4 (CH), 133.5 (C), 148.1 (C),
156.0 (C) ppm.
1
for C42H58N6O6 [M + H]+ 743.4, found 743.3. H-NMR (500 MHz,
CD3OD): δ = 1.10 (s, 6H), 1.65−1.90 (m, 9H), 2.0−2.25 (m, 9H),
2.64 (m, 2H), 2.73 (s, 3H), 2.80 (m, 1H), 2.94 (s, 3H), 2.97 (s, 3H),
3.12 (m, 4H), 3.27 (m, 2H), 3.68 (s, 6H), 6.57 (d, J = 8.5 Hz, 2H), 6.79
(s, 1H), 7.28 (t, J = 8.5 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H), 7.44 (s, 1H),
7.57 (s, 1H) ppm. 13C-NMR (126 MHz, CD3OD): δ = 18.4 (CH3),
23.1 (CH2), 23.9 (CH2), 29.0 (CH2), 29.3 (CH2), 29.9 (CH), 34.5
(CH2), 34.9 (CH2), 35.0 (CH2), 35.8 (CH3), 39.0 (CH2), 39.8 (CH3),
41.2 (CH3), 55.2 (CH3), 57.1 (CH2), 65.5 (CH2), 106.6 (C), 110.8
(CH), 126.2 (CH), 127.1 (CH), 130.2 (CH), 133.8 (C), 138.8 (C),
148.3 (C), 160.7 (C), 176.3 (C) ppm. HRMS: m/z calculated for
C42H58N6O6 [M + H]+ 743.44972, found 743.44937; m/z calculated
for [M + 2H]2+ 372.22883, found 372.22752.
2-((1-(4-((3-((3-(DOTA-methylamino)propyl)methylamino)-
propyl)methylcarbamoyl)-2-isopropylphenyl)-5-(2,6-dimethoxy-
phenyl)-1H-pyrazole-3-carbonyl)amino)adamantane-2-carboxylic
Acid (3BP-227). To a solution of 9 (TFA salt) (17 mg, 19.8 μmol, 1
equiv) in anhydrous DMF (1 mL) and DIPEA (10.3 μL, 59.4 μmol, 3
equiv) was added a solution of DOTA-NHS (30.16 mg, 39.6 μmol, 2
equiv) in anhydrous DMF (1 mL) with DIPEA (41.5 μL, 0.24 mmol, 12
equiv). The reaction mixture was stirred overnight at RT. The reaction
medium was concentrated under vacuum, and the resulting oil was
purified on a BetaBasic-18 column (A: H2O 0.1% TFA, B: MeCN 0.1%
TFA with the following gradient program: 30% of B for 5 min, 30% to
60% of B in 50 min, at a flow rate of 20 mL/min). The product 3BP-227
was obtained as a white powder (4 TFA salt, 22.8 mg, 73%, purity:
99%). RP-HPLC-MS: tr = 3.88 min, m/z calculated for C58H84N10O13
[M + H]+ 1129.6, found 1129.8. HRMS: m/z calculated for
tert-Butyl (3-((3-(Dimethylamino)propyl)amino)propyl)-
carbamate (Synthon C). To a solution of tert-butyl (3-((N-(3-
(dimethylamino)propyl)-2-nitrophenyl)sulfonamido)propyl)-
carbamate (11) (1.1 g, 2.5 mmol, 1 equiv) in DMF (10 mL) was added
cesium carbonate (3.3 g, 10 mmol, 4 equiv). Thiophenol (0.51 mL, 5
mmol, 2 equiv) was then added to the suspension, and the reaction
mixture was stirred at 40 °C for 5 h. The reaction medium was extracted
in a solution of citric acid (pH = 3) and washed three times with
CH2Cl2. After basification with a solution of K2CO3, the compound was
extracted four times with CH2Cl2. The organic layers were combined,
dried over MgSO4, filtered, and concentrated under vacuum. The
yellowish oil was purified by semi-preparative HPLC on a BetaBasic-18
column (A: H2O 0.1% TFA, B: MeCN 0.1% TFA; with the following
gradient program: 0% of B for 10 min, 0% to 40% of B in 50 min, at a
flow rate of 20 mL/min) to obtain a white solid as 3 TFA salt (0.681 g,
56%). RP-HPLC-MS: tr = injection peak, m/z calculated for
1
C13H29N3O2 [M + H]+ 260.2, found 260.3. H-NMR (500 MHz,
DMSO-d6): δ = 1.38 (s, 9H), 1.70 (quint, J = 7.8 Hz, 2H), 1.95 (quint, J
= 7.8 Hz, 2H), 2.78 (s, 6H), 2.89 (m, 2H), 2.96 (m, 2H), 2.99 (m, 2H),
3.11 (m, 2H) ppm; 13C-NMR (126 MHz, DMSO-d6): δ = 21.3 (CH2),
26.7 (CH2), 28.8 (CH3), 37.5 (CH2), 42.7 (CH2), 44.3 (CH3), 45.2
(CH2), 54.2 (CH2), 78.3 (C) ppm (Note: one quaternary carbon not
detected).
Experimental Procedures and Characterization Data for 12.
Ethyl 1-(4-((3-((tert-Butoxycarbonyl)amino)propyl)(3-
(dimethylamino)propyl)carbamoyl)-2-isopropylphenyl)-5-(2,6-di-
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methoxyphenyl)-1H-pyrazole-3-carboxylate (12). To a solution of 11
(0.4 g, 0.91 mmol, 1 equiv) in anhydrous DMF (5 mL) was added
DIPEA (0.174 mL, 1.0 mmol, 1.1 equiv) and HATU (0.38 g, 1.0 mmol,
1.1 equiv). The orange solution was stirred 10 min at RT. A solution of
tert-butyl (3-((3-(dimethylamino)propyl)amino)propyl)carbamate (2
TFA) (synthon C) (0.487 g, 1.0 mmol, 1.1 equiv) in DMF (5 mL) and
DIPEA (0.522 mL, 3.0 mmol, 3.3 equiv) was added to the reaction
mixture. The orange solution was stirred for 6 h at RT. The reaction
mixture was evaporated to obtain an orange oil, which was taken up in
CH2Cl2 and washed twice with brine. The organic layer was dried over
MgSO4, filtered, and concentrated under vacuum. An orange oil was
obtained, which was purified by flash-column chromatography (A:
CH2Cl2, B: MeOH; with the following gradient program: 100% to 70%
of B in 30 CV). The product was isolated as an orange oil (0.602 g, 97%,
purity: 96%). RP-HPLC-MS: tr = 4.27 min, m/z calculated for
196.1, found 196.3. 1H-NMR (500 MHz, DMSO-d6): δ = 1.6−2.2 (m,
14H), 8.59 (br, 2H), 13.84 (br, 1H) ppm; 13C-NMR (126 MHz,
DMSO-d6): δ = 26.2 (CH), 31.2 (CH2), 32.2 (CH), 34.4 (CH2), 37.5
(CH2), 63.9 (C), 171.4 (C) ppm. mp: 271 °C.
tert-Butyl 2-Aminoadamantane-2-carboxylate (Synthon D). A
suspension of 2-aminoadamantane-2-carboxylic acid hydrochloride
(15) (0.7 g, 3.0 mmol, 1 equiv) in tert-butyl acetate (25 mL) was cooled
to 0 °C. HClO4 (0.42 mL, 4.8 mmol, 1.6 equiv) was added dropwise,
and the reaction mixture was stirred at RT overnight. A 2 M NaOH (40
mL) solution was added slowly at 0 °C to quench the reaction until the
pH reached 9−10. The mixture was then extracted with EtOAc (4 ×
100 mL) and washed with brine. After being dried with MgSO4, the
organic layer was filtered and concentrated under vacuum to obtain a
white solid, which was purified by flash-column chromatography (A:
CH2Cl2, B: CH2Cl2/MeOH 9:1; with the following gradient program:
0% to 20% of B in 10 CV, 20% of B for 10 CV). A white solid (0.3 g,
40%, purity: 96%) was obtained. RP-HPLC-MS: tr = 3.7 min, m/z
calculated for C15H25NO2 [M + H]+ 252.2, found 252.2. Rf (CH2Cl2/
MeOH, 98/2) = 0.34. 1H-NMR (500 MHz, CDCl3): δ = 1.35 (m, 2H),
1.40 (s, 9H), 1.47 (m, 2H), 1.60 (m, 2H), 1.69 (m, 6H), 1.97 (m, 2H),
2.15 (m, 2H) ppm; 13C-NMR (126 MHz, CDCl3): δ = 26.9 (CH), 27.2
(CH3), 27.9 (CH), 32.0 (CH2), 34.1 (CH), 35.1 (CH2), 37.8 (CH2),
61.8 (C), 79.9 (C), 176.0 (C) ppm. mp: 99 °C.
1
C37H53N5O7 [M + H]+ 680.4, found 680.5. H-NMR (500 MHz,
CDCl3): δ = 0.98 (s, 6H), 1.39 (s, 9 H), 1.41 (t, J = 7.1 Hz, 3H), 1.68
(m, 2H), 1.81 (m, 1H), 2.04 (m, 1H), 2.19 (m, 2H), 2.72 (m, 1H), 2.79
(s, 6H), 3.1−3.3 (m, 4H), 3.55 (m, 2H), 3.65 (s, 6H), 4.42 (q, J = 7.1
Hz, 2H), 4.81 (br, 1H), 6.46 (d, J = 8.4 Hz, 2H), 6.92 (s, 1H), 7.16 (d, J
= 7.8 Hz, 1H), 7.2−7.25 (m, 2H), 7.39 (d, J = 7.8 Hz, 1H) ppm. 13C-
NMR (126 MHz, CDCl3): δ = 12.2 (CH3), 14.5 (CH3), 17.4 (CH),
18.6 (CH2), 27.6 (CH3), 28.4 (CH3), 42.3 (CH2), 43.1 (CH3), 43.6
(CH3), 54.2 (CH3), 55.5 (CH2), 60.9 (CH2), 81.5 (CH), 103.5 (CH),
111.1 (CH), 124.2 (CH), 128.8 (CH), 131.6 (CH), 133.9 (CH), 158.3
(C), 162.7 (C) ppm.
Experimental Procedures and Characterization Data for
Synthon D. 2-Aminoadamantane-2-carbonitrile (13). A mixture of
2-adamantanone (2 g, 13.3 mmol, 1 equiv), NaCN (0.784 g, 16 mmol,
1.2 equiv), aqueous ammonia 25% solution (2.4 mL), and ammonium
chloride (1.423 g, 26.6 mmol, 2 equiv) was dissolved in water (12 mL)
and EtOH (24 mL). The resulting solution was stirred at 50 °C
overnight. The solution was cooled to RT, extracted with Et2O (120
mL), and concentrated. The white solid obtained was then purified on a
silica plug (heptane/EtOAc 7:3). A white powder (2.16 g, 92%) was
obtained. RP-HPLC-MS: tr = 4.9 min, m/z calculated for C11H16N2 [M
+ H + MeCN]+ 218.1, found 218.1. Rf (heptane/EtOAc, 7/3) = 0.4.
1H-NMR (500 MHz, CDCl3): δ = 1.54 (m, 1H), 1.70 (m, 1H), 1.72
Experimental Procedures and Characterization Data for
Precursor 16. 2-((1-(4-((3-Aminopropyl)-(3-dimethylaminopropyl)-
carbamoyl)-2-isopropylphenyl)-5-(2,6-dimethoxyphenyl)-1H-pyra-
zole-3-carbonyl)amino)adamantane-2-carboxylic acid (16). To a
solution of 12 (0.602 g, 0.86 mmol, 1 equiv) in dioxane (9 mL) was
added a solution of 5 M KOH (0.886 mL, 4.43 mmol, 5 equiv). The
yellow solution was stirred overnight at RT. The reaction mixture was
concentrated under vacuum. The residue was taken up in water and
washed three times with Et2O. The aqueous phase was acidified to pH
3−4 (precipitation) with a solution of 1 M HCl and evaporated. EtOH
was added to the residue and KCl was filtered off. The solution was
concentrated under vacuum to obtain an orange oil. DMF (15 mL),
NEt3 (0.46 mL, 3.3 mmol, 3 equiv), and HATU (0.46 g, 1.2 mmol, 1.1
equiv) were added. The yellowish solution was stirred for 15 min at RT.
A solution of tert-butyl-2-aminoadamantane-2-carboxylate (synthon
D) (0.302 g, 1.2 mmol, 1.1 equiv) in DMF (5 mL) and NEt3 (0.31 mL,
2.2 mmol, 2 equiv) was added to the reaction mixture and stirred for 4 h
at RT. The reaction mixture was concentrated under vacuum to obtain
an orange oil. Finally, CH2Cl2 (5 mL), TFA (5 mL), and H2O (0.5 mL)
were added. The yellow solution was stirred at RT for 1.5 h. The
reaction mixture was concentrated under vacuum and purified by semi-
preparative HPLC on a BetaBasic-18 column (A: H2O 0.1% TFA, B:
MeCN 0.1% TFA with the following gradient program: 20% of b for 5
min, 20% to 60% of B in 50 min, at a flow rate of 20 mL/min) to obtain
16 as a white powder (2 TFA salt, 0.456 g, 55%, purity: 99%). RP-
HPLC-MS: tr = 3.66 min, m/z calculated for C41H56N6O6 [M + H]+
(m, 1H), 1.76 (m, 1H), 1.84 (m, 1H), 1.87 (m, 1H), 1.90 (m, 1H), 1.93
(m, 1H), 1.96 (m, 1H), 1.98 (m, 1H), 2.06 (m, 1H), 2.14 (m, 1H), 2.23
(d, 1H), 2.51 (m, 1H) ppm; 13C-NMR (126 MHz, CDCl3): δ = 26.5
(CH), 26.8 (CH), 27.5 (CH), 30.3 (CH2), 35.0 (CH2), 36.4 (CH2),
36.6 (CH), 37.6 (CH2), 39.3 (CH2), 56.4 (C), 125.5 (C) ppm. mp: 186
°C.
N-(2-Cyanoadamantan-2-yl)benzamide (14). To a solution of 2-
aminoadamantane-2-carbonitrile (13) (1.5 g, 8.5 mmol, 1 equiv) in
THF (15 mL) was added a solution of K2CO3 (1.78 g, 12.9 mmol, 1.52
equiv) in water (15 mL). To the resulting solution was added dropwise,
over 30 min, a solution of benzoyl chloride (1.48 mL, 12.8 mmol, 1.5
equiv) in THF (15 mL) with vigorous stirring at RT. After 5 min, a
white precipitate began to form. After a further 2 h, the white solid was
isolated by filtration and dried under vacuum. The solid was purified on
a silica plug (CH2Cl2/MeOH 95:5) to obtain a white powder (1.64 g,
69%, purity: 90%). RP-HPLC-MS: tr = 4.79 min, m/z calculated for
C18H20N2O [M + H]+ 281.2, found 281.1. Rf (heptane/EtOAc, 7/3) =
0.31. 1H-NMR (500 MHz, CDCl3): δ = 1.76 (m, 3H), 1.85−2.02 (m,
8H), 2.05 (m, 1H), 2.32 (d, 1H), 2.65 (m, 1H), 6.12 (br, 1H), 7.43 (td,
J = 8.5, 1.7 Hz, 2H), 7.52 (tt, J = 8.5, 1.7 Hz, 1H), 7.77 (dt, J = 8.5, 1.7
Hz, 2H) ppm; 13C-NMR (126 MHz, CDCl3): δ = 26.2 (CH), 26.5
(CH), 27.6 (CH2), 31.5 (CH2), 33.9 (CH), 34.2 (CH2), 37.3 (CH2),
39.4 (CH2), 47.1 (CH), 57.3 (C), 120.2 (C), 127.2 (CH), 128.9 (CH),
132.3 (CH), 134.0 (C), 166.6 (C) ppm. mp: 250 °C.
2-Aminoadamantane-2-carboxylic Acid Hydrochloride (15). To
N-(2-cyanoadamantan-2-yl)benzamide (14) (1 g, 3.6 mmol, 1 equiv)
was added acetic acid (18 mL), concentrated HCl (5 mL), and water (3
mL). The white suspension was then heated to reflux for 5 days. The
reaction mixture was concentrated under vacuum to obtain a white
solid, which was triturated twice in hot Et2O (40 mL) and twice in hot
MeCN (25 mL). A white powder (0.768 g, 92%) was obtained. RP-
HPLC-MS: tr = 0.5 min, m/z calculated for C11H17NO2 [M + H]+
1
729.4, found 729.3. H-NMR (500 MHz, CDCl3): δ = 0.97 (s, 6H),
1.49 (m, 2H), 1.63 (m, 4H), 1.67−1.80 (m, 4H), 1.90 (m, 2H), 1.98
(m, 2H), 2.11 (m, 4H), 2.51 (m, 2H), 2.6−2.7 (m, 2H), 2.72 (s, 3H),
2.78 (m, 1H), 2.91 (s, 3H), 3.09 (m, 1H), 3.2−3.35 (m, 3H), 3.57 (s,
6H), 6.6 (d, J = 8.4 Hz, 2H), 6.87 (s, 1H), 7.2−7.4 (m, 4H) ppm. 13C-
NMR (126 MHz, CDCl3): δ = 11.3 (CH3), 26.4 (CH3), 27.6 (CH3),
33.4 (CH2), 36.7 (CH2), 37.0 (CH2), 42.6 (CH3), 42.8 (CH3), 54.5
(CH2), 55.2 (CH2), 55.6 (CH3), 63.6 (CH2), 104.0 (CH), 105.6 (C),
109.0 (CH), 123.5 (CH), 124.3 (CH), 129.9 (CH), 137.8 (C), 158.1
(C), 159.4 (C) ppm. HRMS: m/z calculated for C41H56N6O6 [M + H]+
729.43407, found 729.43468; m/z calculated for [M + Na]+ 751.41590,
found 751.41378.
General Procedure for the Coupling of the Chelating Agent.
To a solution of 16 (1 equiv) in anhydrous DMF (0.5 mL) and DIPEA
(2 equiv) was added a solution of the chelating agent (1.5 equiv) in
anhydrous DMF (0.5 mL) with DIPEA (6−9 equiv depending on the
chelator). The reaction mixture was stirred for 2−4 h at RT until
completion. The reaction medium was concentrated under vacuum,
and the oil was purified on a semi-preparative column.
2-((1-(4-((3-DOTA-aminopropyl)-(3-dimethylaminopropyl)-
carbamoyl)-2-isopropylphenyl)-5-(2,6-dimethoxyphenyl)-1H-pyra-
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zole-3-carbonyl)amino)adamantane-2-carboxylic Acid (3BP-228).
Chelator: DOTA-NHS, Reaction time: 2 h, Equiv. DIPEA: 9.
Purification on a BetaBasic-18 column (A: H2O 0.1% TFA, B:
MeCN 0.1% TFA with the following gradient program: 20% of B for 5
min, 20% to 60% of B in 45 min, at a flow rate of 20 mL/min). The
product 3BP-228 was obtained as a white powder (3 TFA salt, 21 mg,
40%, purity: 93%). RP-HPLC-MS: tr = 3.83 min, m/z calculated for
C57H82N10O13 [M + H]+ 1115.6, found 1115.7. HRMS: m/z calculated
for C57H82N10O13 [M + H]+ 1115.61422, found 1115.61484; m/z
calculated for [M + Na]+ 1137.59605, found 1139.59447.
2-((1-(4-((3-DOTAGA-aminopropyl)-(3-dimethylaminopropyl)-
carbamoyl)-2-isopropylphenyl)-5-(2,6-dimethoxyphenyl)-1H-pyra-
zole-3-carbonyl)amino)adamantane-2-carboxylic Acid (DOTAGA-
16). Chelator: DOTAGA anhydride, Reaction time: 4 h, Equiv. DIPEA:
6. Purification on a BetaBasic-18 column (A: H2O 0.1% TFA, B: MeCN
0.1% TFA with the following gradient program: 30% of B for 5 min,
30% to 70% of B in 50 min, at a flow rate of 20 mL/min). The product
DOTAGA-16 was obtained as a white powder (4 TFA salt, 21.59 mg,
37%, purity: 97%). RP-HPLC-MS: tr = 3.82 min, m/z calculated for
C60H86N10O15 [M + H]+ 1187.6, found 1187.6. HRMS: m/z calculated
for C60H86N10O15 [M + H]+ 1187.63535, found 1187.63587; m/z
calculated for [M + 2H]2+ 594.32165, found 594.32077.
2-((1-(4-((3-NOTA-aminopropyl)-(3-dimethylaminopropyl)-
carbamoyl)-2-isopropylphenyl)-5-(2,6-dimethoxyphenyl)-1H-pyra-
zole-3-carbonyl)amino)adamantane-2-carboxylic Acid (NOTA-16).
Chelator: NOTA-NHS, Reaction time: 3 h, Equiv. DIPEA: 7.5.
Purification on a BetaBasic-18 column (A: H2O 0.1% TFA, B: MeCN
0.1% TFA with the following gradient program: 30% of B for 5 min,
30% to 60% of B in 50 min, at a flow rate of 20 mL/min). The product
NOTA-16 was isolated as a white powder (3 TFA salt, 14.39 mg, 30%,
purity: 98%). RP-HPLC-MS: tr = 3.85 min, m/z calculated for
C53H75N9O11 [M + H]+ 1014.6, found 1014.6. HRMS: m/z calculated
for C53H75N9O11 [M + H]+ 1014.56654, found 1014.56629.
2-((1-(4-((3-NODAGA-aminopropyl)-(3-dimethylaminopropyl)-
carbamoyl)-2-isopropylphenyl)-5-(2,6-dimethoxyphenyl)-1H-pyra-
zole-3-carbonyl)amino)adamantane-2-carboxylic Acid (NODAGA-
16). Chelator: NODAGA-NHS, Reaction time: 2 h, Equiv. DIPEA: 8.
Purification on a BetaBasic-18 column (A: H2O 0.1% TFA, B: MeCN
0.1% TFA with the following gradient program: 30% of B for 5 min,
30% to 70% of B in 50 min, at a flow rate of 20 mL/min). The product
NODAGA-16 was obtained as a white powder (4 TFA salt, 35.89 mg,
65%, purity: 96%). RP-HPLC-MS: tr = 3.9 min, m/z calculated for
C56H79N9O13 [M + H]+ 1086.6, found 1086.7. HRMS: m/z calculated
for C56H79N9O13 [M + H]+ 1086.58767, found 1086.58672; m/z
calculated for [M + 2H]2+ 543.79781, found 543.79634.
2-((1-(4-((3-THP-aminopropyl)-(3-dimethylaminopropyl)-
carbamoyl)-2-isopropylphenyl)-5-(2,6-dimethoxyphenyl)-1H-pyra-
zole-3-carbonyl)amino)adamantane-2-carboxylic Acid (THP-16).
Chelator: THP-NCS, Reaction time: 4 h, Equiv. DIPEA: 9. Purification
on a BetaBasic-18 column (A: H2O 0.1% TFA, B: MeCN 0.1% TFA
with the following gradient program: 30% of B for 5 min, 30% to 60% of
B in 50 min, at a flow rate of 20 mL/min). The product THP-16 was
obtained as a white powder (7 TFA salt, 13.6 mg, 46%, purity: 97%, the
peak at 3.8 min corresponds to the expected compound, and the peak at
4.1 min corresponds to an Fe adduct formed during the analysis). RP-
HPLC-MS: tr = 3.84 min, m/z calculated for C86H112N16O16S2 [M +
2H]2+ 845.4, found 845.7. HRMS: m/z calculated for C56H79N9O13
C86H112N16O16S2 [M + 2H]2+ 845.38630, found 845.40299; m/z
calculated for [M + 3H]3+ 563.93739, found 564.26978.
Experimental Procedures and Characterization Data for
bisDOTAGA-16. Lys(DOTAGA(tBu)4)2. To a solution of DOTAGA-
(tBu)4 (40 mg, 57 μmol, 1.9 equiv) in anhydrous DMF (1 mL) with
DIPEA (21 μL, 120 μmol, 4 equiv) was added HATU (21.7 mg, 57
μmol, 1.9 equiv). The yellow solution was stirred for 15 min at RT, and
DIPEA (21 μL, 120 μmol, 4 equiv) and L-lysine monochlorohydrate
(5.5 mg, 30 mmol, 1 equiv) were added. The reaction mixture was
stirred for 4 h at RT. The reaction medium was concentrated under
vacuum and purified on a BetaBasic-18 column (A: H2O 0.1% TFA, B:
MeCN 0.1% TFA with the following gradient program: 20% of B for 5
min, 20% to 80% of B in 70 min, at a flow rate of 20 mL/min). The
product was obtained as a white powder (4 TFA salt, 25.8 mg, 44%,
purity: 90%). RP-HPLC-MS: tr = 3.88 min, m/z calculated for
C76H138N10O20 [M + 1H]1+ 1512.01, found 1512.1; [M + 2H]2+ 756.5,
found 756.5. HRMS: m/z calculated for C76H138N10O20 [M + H]+
1512.01683, found 1512.01873; m/z calculated for [M + 2H]2+
756.51239, found 756.51434.
BisDOTAGA-16. To a solution of Lys(DOTAGA(tBu)4)2 (15.7 mg,
10.4 μmol, 1 equiv) in anhydrous DMF (0.5 mL) was added DIPEA
(5.4 μL, 31.2 μmol, 3 equiv) and then HATU (3.95 mg, 10.4 μmol, 1
equiv). The solution was stirred 15 min at RT, and a solution of 16
(8.77 mg, 10.4 μmol, 1 equiv) in anhydrous DMF (0.5 mL) with
DIPEA (7.3 μL, 41.6 μmol, 4 equiv) was added. The reaction mixture
was stirred 5 h at RT. The reaction medium was concentrated under
vacuum, and a solution of TFA:triisopropylsilane:H2O (95:2.5:2.5, v/
v/v, 2 mL) was added. The reaction was stirred for 3 h at 45 °C and
concentrated under a flow of nitrogen gas. The solution was purified on
a BetaBasic-18 column (A: H2O 0.1% TFA, B: MeCN 0.1% TFA with
the following gradient program: 20% of B for 5 min, 20% to 60% of B in
60 min, at a flow rate of 20 mL/min). The product bisDOTAGA-16
was obtained as a white powder (4 TFA salt, 7.94 mg, 34%, purity:
97%). RP-HPLC-MS: tr = 3.73 min, m/z calculated for C85H128N16O25
[M + 2H]2+ 887.5, found 888.2; [M + 3H]3+ 592.0, found 592.5.
HRMS: m/z calculated for C85H128N16O25 [M + 2H]2+ 887.46977,
found 887.47499.
Experimental Procedures and Characterization Data for
bisNODAGA-16. Lys(NODAGA(tBu)3)2. To a solution of NODAGA-
(tBu)3 (75 mg, 0.138 mmol, 2.5 equiv) in anhydrous DMF (1 mL) with
DIPEA (47.9 μL, 0.275 mmol, 5 equiv) was added TSTU (50 mg, 0.165
mmol, 3 equiv). The yellow solution was stirred for 30 min at RT, and a
suspension of L-lysine monochlorohydrate (10 mg, 0.055 mmol, 1
equiv) in DMF (1 mL) and DIPEA (28.7 μL, 0.165 mmol, 3 equiv) was
added to the reaction mixture. The yellow suspension was stirred
overnight at RT and heated for 4 h at 50 °C. The reaction medium was
concentrated under vacuum and purified on a BetaBasic-18 column (A:
H2O 0.1% TFA, B: MeCN 0.1% TFA with the following gradient
program: 25% of B for 5 min, 25% to 75% of B in 50 min, at a flow rate of
20 mL/min). The product was obtained as a white powder (3 TFA salt,
66.4 mg, 78%, purity: 92%). RP-HPLC-MS: tr = 4.15 min, m/z
calculated for C60H108N8O16 [M + H]+ 1197.8, found 1197.8; m/z
calculated for [M + 2H]2+ 599.4, found 599.6. HRMS: m/z calculated
for C60H108N8O16 [M + H]+ 1197.79627, found 1197.79853; m/z
calculated for [M + 2H]2+ 599.40211, found 599.40336.
BisNODAGA-16. To a solution of Lys(NODAGA(tBu)3)2 (15 mg,
12.5 μmol, 1 equiv) in anhydrous DMF (0.75 mL) with DIPEA (6.5 μL,
37.5 μmol, 3 equiv) was added HATU (4.75 mg, 12.5 μmol, 1 equiv).
The solution was stirred for 15 min at RT, and a solution of 16 (10.5
mg, 12.5 μmol, 1 equiv) in anhydrous DMF (0.75 mL) with DIPEA
(8.7 μL, 50.0 μmol, 4 equiv) was added. The reaction mixture was left
stirring for 3 h at RT. The reaction medium was concentrated under
vacuum, and a solution of TFA:triisopropylsilane:H2O (95:2.5:2.5, v/
v/v, 2 mL) was added. The reaction was stirred for 6 h at RT and
concentrated under a flow of nitrogen gas. The residue was purified on a
BetaBasic-18 column (A: H2O 0.1% TFA, B: MeCN 0.1% TFA with the
following gradient program: 20% of B for 5 min, 20% to 60% of B in 40
min, at a flow rate of 20 mL/min). The product bisNODAGA-16 was
obtained as a white powder (4 TFA salt, 12.0 mg, 47%, purity: 98%).
RP-HPLC-MS: tr = 3.90 min, m/z calculated for C77H114N14O21 [M +
2H]2+ 786.4, found 786.4. HRMS: m/z calculated for C77H114N14O21
[M + H]+ 1571.83624, found 1571.83830; m/z calculated for [M +
2H]2+ 786.42209, found 786.42406.
General Procedure for the Metalation with natGa. The
compounds were dissolved in acetate buffer (0.5 mL, 0.1 M, pH
3.48). A solution of Ga(NO3)3 (1.5 equiv for 3BP-227, 3BP-228,
DOTAGA-16, NOTA-16, NODAGA-16, and THP-16 and 4 equiv for
bisDOTAGA-16 and bisNODAGA-16) in acetate buffer (0.5 mL) was
then added. The reaction mixture was stirred for 5 h at 40 °C. The
excess of free gallium was removed by semi-preparative RP-HPLC.
natGa-3BP-227. Purification on a BetaBasic-18 column (A: H2O
0.1% TFA, B: MeCN 0.1% TFA with the following gradient program:
30% of B for 5 min, 30% to 60% of B in 50 min, at a flow rate of 20 mL/
min). The product was obtained as a white powder (5 TFA salt, 5.8 mg,
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49%, purity: 97%). RP-HPLC-MS: tr = 3.85 min. MALDI-TOF: m/z
calculated for C58H81GaN10O13 [M + H]+ 1195.532, found 1195.703.
natGa-3BP-228. Purification on a BetaBasic-18 column (A: H2O
0.1% TFA, B: MeCN 0.1% TFA with the following gradient program:
30% of B for 5 min, 30% to 60% of B in 50 min, at a flow rate of 20 mL/
min). The product was obtained as a white powder (3 TFA salt, 8.7 mg,
70%, purity: 97%). RP-HPLC-MS: tr = 3.77 min. MALDI-TOF: m/z
calculated for C57H79GaN10O13 [M + H]+ 1181.516, found 1181.844.
natGa-DOTAGA-16. Purification on a BetaBasic-18 column (A: H2O
0.1% TFA, B: MeCN 0.1% TFA with the following gradient program:
30% of B for 5 min, 30% to 60% of B in 50 min, at a flow rate of 20 mL/
min). The product was obtained as a white powder (5 TFA salt, 7.1 mg,
51%, purity: 92%). RP-HPLC-MS: tr = 3.87 min. MALDI-TOF: m/z
calculated for C60H82GaN10O15− [M + H]+ 1252.530, found 1253.844.
natGa-NOTA-16. Purification on a BetaBasic-18 column (A: H2O
0.1% TFA, B: MeCN 0.1% TFA with the following gradient program:
25% of B for 5 min, 25% to 50% of B in 50 min, at a flow rate of 20 mL/
min). The product was obtained as a white powder (3 TFA salt, 10.6
mg, 84%, purity: 99%). RP-HPLC-MS: tr = 3.66 min. MALDI-TOF: m/
z calculated for C53H73GaN9O11+ [M + H]+ 1081.476, found 1080.467.
natGa-NODAGA-16. Purification on a BetaBasic-18 column (A: H2O
0.1% TFA, B: MeCN 0.1% TFA with the following gradient program:
30% of B for 5 min, 30% to 60% of B in 50 min, at a flow rate of 20 mL/
min). The compound was obtained as a white powder (3 TFA salt, 4.35
mg, 35%, purity >99%). RP-HPLC-MS: tr = 3.9 min. MALDI-TOF: m/
z calculated for C56H76GaN9O13 [M + H]+ 1152.490, found 1152.454.
natGa-THP-16. Purification on a BetaBasic-18 column (A: H2O 0.1%
TFA, B: MeCN 0.1% TFA with the following gradient program: 35% of
B for 5 min, 35% to 60% of B in 40 min, at a flow rate of 20 mL/min).
The product was obtained as a white powder (9 TFA salt, 6 mg, 53%,
purity: 77 + 20%). RP-HPLC-MS: tr = 4.2 min. MALDI-TOF: m/z
calculated for C86H109GaN16O16S2 [M + H]+ 1755.698, found
1757.551.
Radiolabeling of Compounds with 68Ga. Compounds were
radiolabeled using a Modular-Lab PharmTracer (Eckert & Ziegler).
Fractional elution of the 68Ge/68Ga generator gave a solution (1.5 mL,
90 MBq), which was added to a solution of 2.99 nmol of each
compound in EtOH (150 μL) and ammonium acetate buffer (1 M, pH
6.88) to adjust the pH to 3.5. The reaction mixture was incubated at 37
°C for 5 min for the NOTA-, NODAGA-, and THP-based compounds,
and at 95 °C for 10 min for the DOTA- and DOTAGA-based
compounds. The products were purified on a C18 Sep-Pack cartridge
(Waters, Milford, MA), eluted using 1000 μL of 80% ethanol in the
second reactor, and subsequently evaporated at 70 °C for 10 min under
an air flow. The final products were diluted with 0.9% sodium chloride
(NaCl). HPLC quality control was performed using a HPLC system
LC-2000 analytical series (Jasco) equipped with a Flowcount
radioactivity detector (Bioscan). Separation was achieved using an
RP C18 Kinetex column (Phenomenex) (2.6 μm, 100 Å, 50 × 2.1 mm)
with ultrapure water and HPLC-grade MeCN (A: H2O 0.1% TFA and
B: MeCN 0.1% TFA). Analyses were performed with the following
gradient program: 5% to 95% of B in 5 min, 95% B for 1.5 min, at a flow
rate of 0.5 mL/min. The quantity of colloids was evaluated using a
system of two instant thin layer chromatographies (iTLC) eluted with
citrate sodium and NH4Ac/MeOH as mobile phases.
Small Animal PET Imaging and Biodistribution Studies on
the HT29 Tumor Model. All animal studies were conducted at and
approved by the Centre George François Leclerc (CGFL) in
accordance with the relevant guidelines and legislation on the use of
laboratory animals (directive 2010/63/EU) and were approved by the
accredited Ethical Committee (C2ea Grand Campus no. 105) and
French Ministry of Higher Education and Research (#15316). HT-29
tumor bearing mice (8 weeks old, 10 × 106 cells per mouse injected
subcutaneously in the right flack) were intravenously injected in the tail
vein with 3−8 MBq of each radiotracer based on a consistent number of
picomoles being injected (500 pmol). Static PET and MRI images were
simultaneously recorded 90 min p.i. for 30 min. The mice were
awakened between the administration of the tracer and imaging times.
Images were recorded in a dual-ring SiPM microPET fully integrated in
a 7T preclinical MRI (MR Solutions, Guilford, UK). The animals were
kept under anesthesia using 2% isofluorane in oxygen and positioned in
a dedicated heating cradle during imaging. Respiratory gating was
performed with abdominal pressure sensor and dedicated software (PC
Sam, SAII, Stony Brook, US). List-mode data were collected in the PET
system during 30 min. Coronal T1 weighted fast spin echo MR images
were acquired with respiratory gating and the following parameters: 16
slices with a time of repetition (TR) of 1000 ms, time of echo (TE) of
11 ms, 3 signal averages, 1 mm slice thickness, field of view (FOV) of 70
mm, and 256 × 256 pixel matrix. Images were reconstructed with the
3D ordered subset expectation maximization (OSEM) algorithm
implemented in the system using 2 iterations, 32 subsets, and an
isotropic voxel size of 0.28 mm. An energy window of 250−750 keV and
a coincidence time window of 8 ns were applied to the list-mode data.
The algorithm takes into account normalization, random, and decay
corrections. No attenuation correction was applied. Two hours post-
injection, mice were sacrificed and the main organs and tissues were
dissected, weighed, and γ-counted (1480 Wizard 3, PerkinElmer).
Blocking experiments were performed by co-injecting an excess (100
equivalents) of SR142948A. Tumor or tissue uptake are expressed as
mean SD percentage injected dose per gram (%ID/g), corrected for
radionuclide decay. Statistical significance between different groups was
analyzed by the one-way analysis of variance, with [68Ga]Ga-3BP-228
as a control group, followed by Bonferroni correction. Statistical
significance in blocking experiments was assessed by the unpaired two-
tailed Student’s t-test.
natGa-bisDOTAGA-16. Purification on a BetaBasic-18 column (A:
H2O 0.1% TFA, B: MeCN 0.1% TFA with the following gradient
program: 20% of B for 5 min, 20% to 60% of B in 40 min, at a flow rate of
20 mL/min). The product was obtained as a white powder (3 TFA salt,
5.44 mg, 78%, purity: 97.4%). RP-HPLC-MS: tr = 3.73 min. MALDI-
TOF: m/z calculated for C85H122Ga2N16O25 [M + H]+ 1907.735, found
1908.386.
natGa-bisNODAGA-16. Purification on a BetaBasic-18 column (A:
H2O 0.1% TFA, B: MeCN 0.1% TFA with the following gradient
program: 20% of B for 5 min, 20% to 60% of B in 40 min, at a flow rate of
20 mL/min). The product was obtained as a white powder (3 TFA salt,
5.33 mg, 77%, purity: 96%). RP-HPLC-MS: tr = 3.72 min. MALDI-
TOF: m/z calculated for C77H108Ga2N14O21 [M + H]+ 1706.643, found
1706.394.
In Vitro Stability. Fifteen microliters of a 5 mM stock solution of
compound in DMSO 50% (v/v) was stirred in a thermomixer (900
rpm, 37 °C, dark) with 135 μL of mouse serum (Sigma Aldrich, M5905,
batch SLBT4412). The final concentration in peptide was 0.5 mM.
After 0 min, 30 min, 1 h, 2 h, 4 h, and 24 h, a sample (15 μL) of solution
was collected and diluted with 60 μL of ethanol 99%. Fifteen microliters
of a 1 mM stock solution of Fmoc-Trp(tBu)-OH was added to the
suspension for internal calibration. Samples were vortexed and
centrifuged (10,000 rpm, 10 min) to remove precipitated proteins.
The supernatant was analyzed by RP-HPLC-MS at a wavelength of 260
nm.
In Vitro Binding Affinity. The affinity of each compound Ga-3BP-
227, Ga-3BP-228, Ga-DOTAGA-16, Ga-NOTA-16, Ga-NODAGA-
16, Ga-THP-16, Ga-bisDOTAGA-16, and Ga-bisNODAGA-16 for
NTS1 was determined on a competitive binding assay on human
recombinant CHO cells overexpressing NTS1, using [125I]I-Tyr3-
neurotensin (0.05 nM; Kd = 0.22 nM) as the radioligand (Eurofins,
France). Non-specific binding was measured in the presence of an
excess of neurotensin (1 μM). Experiments were performed in
duplicate. Data were fitted by non-linear regression, using GraphPad
Prism 6.0 software. The minimum was set to 0. IC50 values were
converted into inhibition constants (Ki) using the Cheng−Prusoff
equation.
Binding Studies on HT29 Cells. Experiments were performed
with 1 × 106 cells per well in 24-well plates. Cells were washed with ice
cold PBS twice before their incubation with 8 different concentrations
of [68Ga]Ga-bisNODAGA-16 (from 78 pM to 10 nM) in 1 mL of
incubation medium. The compound was incubated alone or in the
presence of an excess (1 μM) of the ligands SR142948A, neurotensin,
or levocabastine, for 60 min at 4 °C. The medium was then withdrawn,
cells were washed twice with ice-cold PBS and solubilized in 1 M
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NaOH. The amount of radioactivity was measured using a γ-counter
(Wallac Wizard 1470, Perkin Elmer, USA). Experiments were
performed in triplicate. Data were fitted, using GraphPad Prism 6.0
software, by applying the one site binding model.
Calcium Mobilization Assay. Antagonist activities were deter-
mined on a fluorescence assay measuring [Ca2+] mobilization in CHO
cells overexpressing human recombinant NTS1, upon stimulation by
neurotensin (0.3 nM), in the presence of increasing concentrations of
natGa-bisNODAGA-16 (Eurofins, France).19 SR142948A was used as
an internal control (IC50 = 23 nM; KB = 2.1 nM). Results are expressed
as a percent inhibition of control agonist response. IC50 values were
determined by non-linear regression of normalized response curves,
using GraphPad Prism 6.0 software.
Mathieu Moreau − Institut de Chimie Moléculaire de
l’Université de Bourgogne, ICMUB UMR CNRS 6302,
Université Bourgogne Franche-Comté, Dijon 21000, France
Pierre-Simon Bellaye − Georges-Franco
Center - UNICANCER, Dijon 21000, France
Mélanie Guillemin − Georges-Francois LECLERC Cancer
Center - UNICANCER, Dijon 21000, France
is LECLERC Cancer Center -
̧ is LECLERC Cancer
̧
Bertrand Collin − Georges-Franco
̧
UNICANCER, Dijon 21000, France
Aurélie Prignon − UMS28 Laboratoire d’Imagerie Moléculaire
Positonique (LIMP), Sorbonne Université, Paris 75020,
France; orcid.org/0000-0002-3963-863X
Small Animal PET-CT Imaging and Biodistribution Studies
on the AsPC-1 Tumor Model. Animal experiments were conducted
in compliance with the French laws, in application of the directive
2010/63/ EU and were approved by the “Charles Darwin” Institutional
Animal Care and Use Committee of Sorbonne University #13184.
AsPC1 tumor bearing mice (10 weeks old, 5 × 106 cells per mouse
injected subcutaneously on the right shoulder in a 1:1 mixture of PBS
and matrigel (BD Biosciences)) were intravenously (retro-orbitary
sinus) injected with 4−6 MBq of a radiotracer (619 36 pmol). Whole
body static PET/computed tomography (CT) scans were performed
under general anesthesia in a NanoScan PET/CT (Mediso Medical
Imaging Systems Ltd., Budapest, Hungary) 50 or 90 min after injection
during 20 min scans using multiple bed (three mice simultaneously).
Anesthesia was induced and maintained during imaging by the
administration of a mixture of isoflurane (1.5−2.5%) and oxygen. CT
acquisitions were performed before PET acquisitions using the same
bed position. PET and CT files were fused and converted to
standardized uptake value (SUV) images using Nucline 2.03 Software
(Mediso Medical Imaging Systems, Hungary). Images are analyzed and
presented as coronal or axial slices. Ex vivo biodistribution was
measured for n = 4 mice at 120 min post-injection (p.i.). Organs were
collected, weighed, and γ-counted (Packard Instrument). Tumor or
tissue uptakes are expressed as mean SD percentage injected dose per
gram (%ID/g), corrected for radionuclide decay.
Franck Denat − Institut de Chimie Moléculaire de l’Université
de Bourgogne, ICMUB UMR CNRS 6302, Université
Bourgogne Franche-Comté, Dijon 21000, France
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00523
Author Contributions
All authors have given approval to the final version of the
manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank the Plateforme d’Analyse Chimique et de
̀
Synthese Moléculaire de l’Université de Bourgogne (http://
www.wpcm.fr) for access to analytical instrumentation (spe-
cially Dr. M. Laly for the ionic chromatography and Dr. Q.
Bonnin for the HRMS analysis). This work was partly funded by
the French National Research Agency (ANR) under the French
Investissements d’Avenir programs Equipex (IMAPPI, ANR-10-
EQPX-05-01), AAP Générique 2017 (ZINELABEL, ANR-17-
CE18-0016), and France Life Imaging (ANR-11-INBS-0006).
This work was also supported by the CNRS and the Université
de Bourgogne. This work is part of the project Pharmaco-
imagerie et agents théranostiques funded by the Conseil
Régional de Bourgogne Franche-Comté through the Plan
d’Action Régional pour l’Innovation (PARI) and by the
European Union through the PO FEDER-FSE 2014/2020
Bourgogne program.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00523.
Materials and general procedures, synthesis schemes, and
analytical data for all compounds, in vitro stability data, in
vitro binding affinity data, radiolabeling data, PET-MRI
images, PET-CT images, tables of ex vivo biodistribution
data determined by γ-counting, and blocking experiment
data (PDF)
ABBREVIATIONS
■
CHO, Chinese hamster ovary; DOTA, 1,4,7,10-tetraazacyclo-
dodecane-1,4,7,10-tetraacetic acid; DOTAGA, 1,4,7,10-tetraa-
zacyclododecane-1-glutaric acid-4,7,10-triacetic acid; NCS, N-
chlorosuccinimide; NHS, N-hydroxysuccinimide; NOTA, 1,4,7-
triazacyclononane-1,4,7-triacetic acid; NODAGA, 1,4,7-triaza-
cyclononane-1-glutaric acid-4,7-diacetic acid; NTS1, neuro-
tensin receptor 1; PBS, phosphate-buffered saline; PET,
positron emission tomography; RP-HPLC, reversed-phase
high-performance liquid chromatography; RT, room temper-
ature; SD, standard deviation
Animated 3D PET images (MP4)
SMILES formula of each compound and associated
biochemical data (CSV)
AUTHOR INFORMATION
■
Corresponding Author
Victor Goncalves − Institut de Chimie Moléculaire de
l’Université de Bourgogne, ICMUB UMR CNRS 6302,
Université Bourgogne Franche-Comté, Dijon 21000, France;
orcid.org/0000-0001-9854-4409;
REFERENCES
■
(1) Vaquero, J. J.; Kinahan, P. Positron Emission Tomography:
Current Challenges and Opportunities for Technological Advances in
Clinical and Preclinical Imaging Systems. Annu. Rev. Biomed. Eng. 2015,
17, 385−414.
(2) Wu, Z.; Martinez-Fong, D.; Trédaniel, J.; Forgez, P. Neurotensin
and Its High Affinity Receptor 1 as a Potential Pharmacological Target
in Cancer Therapy. Front. Endocrinol. 2013, 3, 184.
Email: victor.goncalves@u-bourgogne.fr
Authors
Emma Renard − Institut de Chimie Moléculaire de l’Université
de Bourgogne, ICMUB UMR CNRS 6302, Université
Bourgogne Franche-Comté, Dijon 21000, France
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(3) Carraway, R. E.; Plona, A. M. Involvement of Neurotensin in
Cancer Growth: Evidence, Mechanisms and Development of
Diagnostic Tools. Peptides 2006, 27, 2445−2460.
(21) Lang, C.; Maschauer, S.; Hu
Synthesis and Evaluation of a
̈
bner, H.; Gmeiner, P.; Prante, O.
F-Labeled Diarylpyrazole Glyco-
18
conjugate for the Imaging of NTS1-Positive Tumors. J. Med. Chem.
2013, 56, 9361−9365.
(4) Reubi, J. C.; Waser, B.; Friess, H.; Buchler, M.; Laissue, J.
Neurotensin Receptors: A New Marker for Human Ductal Pancreatic
Adenocarcinoma. Gut 1998, 42, 546−550.
(22) Schulz, J.; Rohracker, M.; Stiebler, M.; Goldschmidt, J.; Stöber,
F.; Noriega, M.; Pethe, A.; Lukas, M.; Osterkamp, F.; Reineke, U.;
Höhne, A.; Smerling, C.; Amthauer, H. Proof of Therapeutic Efficacy of
(5) Seethalakshmi, L.; Mitra, S. P.; Dobner, P. R.; Menon, M.;
Carraway, R. E. Neurotensin Receptor Expression in Prostate Cancer
Cell Line and Growth Effect of NT at Physiological Concentrations.
The Prostate 1997, 31, 183−192.
(6) Reubi, J. C.; Waser, B.; Schaer, J. C.; Laissue, J. A. Neurotensin
Receptors in Human Neoplasms: High Incidence in Ewing’s Sarcomas.
Int. J. Cancer 1999, 82, 213−218.
(7) Maoret, J. J.; Pospaï, D.; Rouyer-Fessard, C.; Couvineau, A.;
Laboisse, C.; Voisin, T.; Laburthe, M. Neurotensin Receptor and Its
MRNA Are Expressed in Many Human Colon Cancer Cell Lines but
Not in Normal Colonic Epithelium: Binding Studies and RT-PCR
Experiments. Biochem. Biophys. Res. Commun. 1994, 203, 465−471.
(8) Souazé, F.; Dupouy, S.; Viardot-Foucault, V.; Bruyneel, E.; Attoub,
S.; Gespach, C.; Gompel, A.; Forgez, P. Expression of Neurotensin and
NT1 Receptor in Human Breast Cancer: A Potential Role in Tumor
Progression. Cancer Res. 2006, 66, 6243−6249.
(9) Reeve, J. G.; Goedert, M.; Emson, P. C.; Bleehen, N. M.
Neurotensin in Human Small Cell Lung Carcinoma. Recent Results
Cancer Res. 1985, 99, 175−176.
(10) Carraway, R.; Leeman, S. E. The Isolation of a New Hypotensive
Peptide, Neurotensin, from Bovine Hypothalami. J. Biol. Chem. 1973,
248, 6854−6861.
(11) Prignon, A.; Provost, C.; Alshoukr, F.; Wendum, D.; Couvelard,
A.; Barbet, J.; Forgez, P.; Talbot, J.-N.; Gruaz-Guyon, A. Preclinical
Evaluation of 68Ga-DOTA-NT-20.3: A Promising PET Imaging Probe
To Discriminate Human Pancreatic Ductal Adenocarcinoma from
Pancreatitis. Mol. Pharmaceutics 2019, 16, 2776−2784.
(12) Maschauer, S.; Prante, O. Radiopharmaceuticals for Imaging and
Endoradiotherapy of Neurotensin Receptor-Positive Tumors. J.
Labelled Compd. Radiopharm. 2018, 61, 309−325.
177
a
Lu-Labeled Neurotensin Receptor 1 Antagonist in a Colon
Carcinoma Xenograft Model. J. Nucl. Med. 2017, 58, 936−941.
(23) Ginj, M.; Zhang, H.; Waser, B.; Cescato, R.; Wild, D.; Wang, X.;
Erchegyi, J.; Rivier, J.; Mäcke, H. R.; Reubi, J. C. Radiolabeled
Somatostatin Receptor Antagonists Are Preferable to Agonists for in
Vivo Peptide Receptor Targeting of Tumors. Proc. Natl. Acad. Sci. U. S.
A. 2006, 103, 16436−16441.
(24) Fani, M.; Maecke, H. R. Radiopharmaceutical Development of
Radiolabelled Peptides. Eur. J. Nucl. Med. Mol. Imaging 2012, 39, 11−
30.
(25) Dash, A.; Chakraborty, S.; Pillai, M. R. A.; Knapp, F. F. R., Jr.
Peptide Receptor Radionuclide Therapy: An Overview. Cancer Biother.
Radiopharm. 2015, 30, 47−71.
(26) Schulz, J.; Rohracker, M.; Stiebler, M.; Goldschmidt, J.; Grosser,
O. S.; Osterkamp, F.; Pethe, A.; Reineke, U.; Smerling, C.; Amthauer,
H. Comparative Evaluation of the Biodistribution Profiles of a Series of
Nonpeptidic Neurotensin Receptor-1 Antagonists Reveals a Promising
Candidate for Theranostic Applications. J. Nucl. Med. 2016, 57, 1120−
1123.
(27) Study to Evaluate the Safety and Activity (Including Distribution) of
177Lu-3BP-227 in Subjects With Solid Tumours Expressing Neurotensin
Receptor Type 1. - Tabular View - ClinicalTrials.gov. https://clinicaltrials.
gov/ct2/show/record/NCT03525392, (accessed Apr 23, 2020).
(28) Baum, R. P.; Singh, A.; Schuchardt, C.; Kulkarni, H. R.; Klette, I.;
Wiessalla, S.; Osterkamp, F.; Reineke, U.; Smerling, C. 177 Lu-3BP-227
for Neurotensin Receptor 1−Targeted Therapy of Metastatic
Pancreatic Adenocarcinoma: First Clinical Results. J. Nucl. Med.
2018, 59, 809−814.
(29) Ametamey, S. M.; Honer, M.; Schubiger, P. A. Molecular
Imaging with PET. Chem. Rev. 2008, 108, 1501−1516.
(13) Yin, X.; Wang, M.; Wang, H.; Deng, H.; He, T.; Tan, Y.; Zhu, Z.;
Wu, Z.; Hu, S.; Li, Z. Evaluation of Neurotensin Receptor 1 as a
Potential Imaging Target in Pancreatic Ductal Adenocarcinoma. Amino
Acids 2017, 49, 1325−1335.
(30) Brandt, M.; Cardinale, J.; Aulsebrook, M. L.; Gasser, G.; Mindt,
T. L. An Overview of PET Radiochemistry, Part 2: Radiometals. J. Nucl.
Med. 2018, 59, 1500−1506.
(31) International Atomic Energy Agency. Gallium-68 Cyclotron
Production; TECDOC Series; International Atomic Energy Agency:
Vienna, 2019.
(14) Renard, E.; Dancer, P.-A.; Portal, C.; Denat, F.; Prignon, A.;
Goncalves, V. Design of Bimodal Ligands of Neurotensin Receptor 1
for Positron Emission Tomography Imaging and Fluorescence-Guided
Surgery of Pancreatic Cancer. J. Med. Chem. 2020, 63, 2426−2433.
(32) Price, E. W.; Orvig, C. Matching Chelators to Radiometals for
Radiopharmaceuticals. Chem. Soc. Rev. 2014, 43, 260−290.
(33) Spang, P.; Herrmann, C.; Roesch, F. Bifunctional Gallium-68
Chelators: Past, Present, and Future. Seminars in Nuclear Medicine
2016, 46, 373−394.
(34) Tsionou, M. I.; Knapp, C. E.; Foley, C. A.; Munteanu, C. R.;
Cakebread, A.; Imberti, C.; Eykyn, T. R.; Young, J. D.; Paterson, B. M.;
Blower, P. J.; Ma, M. T. Comparison of Macrocyclic and Acyclic
Chelators for Gallium-68 Radiolabelling. RSC Adv. 2017, 7, 49586−
49599.
(35) Malmberg, J.; Perols, A.; Varasteh, Z.; Altai, M.; Braun, A.;
Sandström, M.; Garske, U.; Tolmachev, V.; Orlova, A.; Karlström, A. E.
Comparative Evaluation of Synthetic Anti-HER2 Affibody Molecules
Site-Specifically Labelled with 111In Using N-Terminal DOTA,
NOTA and NODAGA Chelators in Mice Bearing Prostate Cancer
Xenografts. Eur. J. Nucl. Med. Mol. Imaging 2012, 39, 481−492.
(36) Mitran, B.; Varasteh, Z.; Selvaraju, R. K.; Lindeberg, G.;
Sörensen, J.; Larhed, M.; Tolmachev, V.; Rosenström, U.; Orlova, A.
Selection of Optimal Chelator Improves the Contrast of GRPR Imaging
Using Bombesin Analogue RM26. Int. J. Oncol. 2016, 48, 2124−2134.
(37) Mitran, B.; Thisgaard, H.; Rinne, S.; Dam, J. H.; Azami, F.;
Tolmachev, V.; Orlova, A.; Rosenström, U. Selection of an Optimal
Macrocyclic Chelator Improves the Imaging of Prostate Cancer Using
Cobalt-Labeled GRPR Antagonist RM26. Sci. Rep. 2019, 9, 17086.
(38) Rinne, S. S.; Dahlsson Leitao, C.; Gentry, J.; Mitran, B.;
Abouzayed, A.; Tolmachev, V.; Ståhl, S.; Löfblom, J.; Orlova, A.
̀
(15) Kitabgi, P.; De Nadai, F.; Rovere, C.; Bidard, J.-N. Biosynthesis,
Maturation, Release, and Degradation of Neurotensin and Neuromedin
N. Ann. N. Y. Acad. Sci. 1992, 668, 30−42.
(16) Gabriel, M.; Decristoforo, C.; Wöll, E.; Eisterer, W.; Nock, B.;
Maina, T.; Moncayo, R.; Virgolini, I. [99mTc]Demotensin VI:
Biodistribution and Initial Clinical Results in Tumor Patients of a
Pilot/Phase I Study. Cancer Biother. Radiopharm. 2011, 26, 557−563.
(17) Gully, D.; Canton, M.; Boigegrain, R.; Jeanjean, F.; Molimard, J.
C.; Poncelet, M.; Gueudet, C.; Heaulme, M.; Leyris, R.; Brouard, A.
Biochemical and Pharmacological Profile of a Potent and Selective
Nonpeptide Antagonist of the Neurotensin Receptor. Proc. Natl. Acad.
Sci. U. S. A. 1993, 90, 65−69.
(18) Myers, R. M.; Shearman, J. W.; Kitching, M. O.; Ramos-
Montoya, A.; Neal, D. E.; Ley, S. V. Cancer, Chemistry, and the Cell:
Molecules That Interact with the Neurotensin Receptors. ACS Chem.
Biol. 2009, 4, 503−525.
(19) Gully, D.; Labeeuw, B.; Boigegrain, R.; Oury-Donat, F.; Bachy,
A.; Poncelet, M.; Pecceu, F.; Maffrand, J. P. Biochemical and
Pharmacological Activities of SR 142948A, a New Potent Neurotensin
Receptor Antagonist. J. Pharmacol. Exp. Ther. 1997, 280, 802−812.
(20) Betancur, C.; Canton, M.; Burgos, A.; Labeeuw, B.; Gully, D.;
Rostene, W.; Pelaprata, D. Characterization of Binding Sites of a New
Neurotensin Receptor Antagonist, [3H]SR 142948A, in the Rat Brain.
Eur. J. Pharmacol. 1998, 343, 67−77.
8577
https://doi.org/10.1021/acs.jmedchem.1c00523
J. Med. Chem. 2021, 64, 8564−8578

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Increase in Negative Charge of 68Ga/Chelator Complex Reduces
Unspecific Hepatic Uptake but Does Not Improve Imaging Properties
of HER3-Targeting Affibody Molecules. Sci. Rep. 2019, 9, 17710.
(39) Rinne, S. S.; Leitao, C. D.; Mitran, B.; Bass, T. Z.; Andersson, K.
G.; Tolmachev, V.; Ståhl, S.; Löfblom, J.; Orlova, A. Optimization of
HER3 Expression Imaging Using Affibody Molecules: Influence of
Chelator for Labeling with Indium-111. Sci. Rep. 2019, 9, 655.
(40) Varasteh, Z.; Mitran, B.; Rosenström, U.; Velikyan, I.; Rosestedt,
M.; Lindeberg, G.; Sörensen, J.; Larhed, M.; Tolmachev, V.; Orlova, A.
The Effect of Macrocyclic Chelators on the Targeting Properties of the
68Ga-Labeled Gastrin Releasing Peptide Receptor Antagonist PEG2-
RM26. Nucl. Med. Biol. 2015, 42, 446−454.
Delaloye, A. Radiolabeled Neurotensin Analog, 99mTc-NT-XI,
Evaluated in Ductal Pancreatic Adenocarcinoma Patients. J. Nucl.
Med. 2003, 44, 1649−1654.
(56) Fani, M.; Del Pozzo, L.; Abiraj, K.; Mansi, R.; Tamma, M. L.;
Cescato, R.; Waser, B.; Weber, W. A.; Reubi, J. C.; Maecke, H. R. PET
of Somatostatin Receptor-Positive Tumors Using 64Cu- and 68Ga-
Somatostatin Antagonists: The Chelate Makes the Difference. J. Nucl.
Med. 2011, 52, 1110−1118.
(57) Banerjee, S. R.; Chen, Z.; Pullambhatla, M.; Lisok, A.; Chen, J.;
Mease, R. C.; Pomper, M. G. Preclinical Comparative Study of 68Ga-
Labeled DOTA, NOTA, and HBED-CC Chelated Radiotracers for
Targeting PSMA. Bioconjugate Chem. 2016, 27, 1447−1455.
(58) Roosenburg, S.; Laverman, P.; Joosten, L.; Cooper, M. S.;
Kolenc-Peitl, P. K.; Foster, J. M.; Hudson, C.; Leyton, J.; Burnet, J.;
Oyen, W. J. G.; Blower, P. J.; Mather, S. J.; Boerman, O. C.; Sosabowski,
J. K. PET and SPECT Imaging of a Radiolabeled Minigastrin Analogue
Conjugated with DOTA, NOTA, and NODAGA and Labeled with
(64)Cu, (68)Ga, and (111)In. Mol. Pharmaceutics 2014, 11, 3930−
3937.
(59) Leitao, C. D.; Rinne, S. S.; Mitran, B.; Vorobyeva, A.; Andersson,
K. G.; Tolmachev, V.; Ståhl, S.; Löfblom, J.; Orlova, A. Molecular
Design of HER3-Targeting Affibody Molecules: Influence of Chelator
and Presence of HEHEHE-Tag on Biodistribution of 68Ga-Labeled
Tracers. Int. J. Mol. Sci. 2019, 20, 1080.
(60) von Witting, E.; Garousi, J.; Lindbo, S.; Vorobyeva, A.; Altai, M.;
Oroujeni, M.; Mitran, B.; Orlova, A.; Hober, S.; Tolmachev, V.
Selection of the Optimal Macrocyclic Chelators for Labeling with 111In
and 68Ga Improves Contrast of HER2 Imaging Using Engineered
Scaffold Protein ADAPT6. Eur. J. Pharm. Biopharm. 2019, 140, 109−
120.
(61) Lin, M.; Welch, M. J.; Lapi, S. E. Effects of Chelator
Modifications on 68Ga-Labeled [Tyr3]Octreotide Conjugates. Mol.
Imaging Biol. 2013, 15, 606−613.
(62) Young, J. D.; Abbate, V.; Imberti, C.; Meszaros, L. K.; Ma, M. T.;
Terry, S. Y. A.; Hider, R. C.; Mullen, G. E.; Blower, P. J. 68Ga-THP-
PSMA: A PET Imaging Agent for Prostate Cancer Offering Rapid,
Room-Temperature, 1-Step Kit-Based Radiolabeling. J. Nucl. Med.
2017, 58, 1270−1277.
(63) Fanelli, R.; Chastel, A.; Previti, S.; Hindié, E.; Vimont, D.;
Zanotti-Fregonara, P.; Fernandez, P.; Garrigue, P.; Lamare, F.;
Schollhammer, R.; Balasse, L.; Guillet, B.; Rémond, E.; Morgat, C.;
Cavelier, F. Silicon-Containing Neurotensin Analogues as Radiophar-
maceuticals for NTS(1)-Positive Tumors Imaging. Bioconjugate Chem.
2020, 31, 2339−2349.
́
(41) Roxin, A.; Zhang, C.; Huh, S.; Lepage, M.; Zhang, Z.; Lin, K.-S.;
18
Bénard, F.; Perrin, D. M. A Metal-Free DOTA-Conjugated
F-
Labeled Radiotracer: [ 18 F]DOTA-AMBF 3 -LLP2A for Imaging VLA-
4 Over-Expression in Murine Melanoma with Improved Tumor Uptake
and Greatly Enhanced Renal Clearance. Bioconjugate Chem. 2019, 30,
1210−1219.
(42) Labeeuw, B.; Gully, D.; Jeanjean, F.; Molimard, J.-C.; Boigegrain,
R. Manufacturing method of semiconductor device. Patent
JP5723483B2. 1998, 58.
(43) Baxendale, I. R.; Cheung, S.; Kitching, M. O.; Ley, S. V.;
Shearman, J. W. The Synthesis of Neurotensin Antagonist SR 48692 for
Prostate Cancer Research. Bioorg. Med. Chem. 2013, 21, 4378−4387.
(44) Kurosawa, W.; Kan, T.; Fukuyama, T. Preparation of Secondary
Amines from Primary Amines via 2-Nitrobenzenesulfonamides: N-(4-
Methoxybenzyl)-3-Phenylpropylamine. Org. Synth. 2002, 79, 186.
(45) Battilocchio, C.; Baxendale, I. R.; Biava, M.; Kitching, M. O.; Ley,
S. V. A Flow-Based Synthesis of 2-Aminoadamantane-2-Carboxylic
Acid. Org. Process Res. Dev. 2012, 16, 798−810.
(46) Battilocchio, C.; Deadman, B. J.; Nikbin, N.; Kitching, M. O.;
Baxendale, I. R.; Ley, S. V. A Machine-Assisted Flow Synthesis of
SR48692: A Probe for the Investigation of Neurotensin Receptor-1.
Chem. − Eur. J. 2013, 19, 7917−7930.
(47) Lang, C.; Gmeiner, P. Efficient Synthesis of Heterocyclic
Neurotensin Receptor Ligands by Microwave-Assisted Aminocarbo-
nylation. Synthesis 2013, 45, 2474−2480.
(48) Price, T. W.; Greenman, J.; Stasiuk, G. J. Current Advances in
Ligand Design for Inorganic Positron Emission Tomography Tracers
68Ga, 64Cu, 89Zr and 44Sc. Dalton Trans. 2016, 45, 15702−15724.
(49) Vita, N.; Laurent, P.; Lefort, S.; Chalon, P.; Dumont, X.; Kaghad,
M.; Gully, D.; Le Fur, G.; Ferrara, P.; Caput, D. Cloning and Expression
of a Complementary DNA Encoding a High Affinity Human
Neurotensin Receptor. FEBS Lett. 1993, 317, 139−142.
̀
(50) Andrés, A.; Rosés, M.; Rafols, C.; Bosch, E.; Espinosa, S.; Segarra,
V.; Huerta, J. M. Setup and Validation of Shake-Flask Procedures for
the Determination of Partition Coefficients (LogD) from Low Drug
Amounts. Eur. J. Pharm. Sci. 2015, 76, 181−191.
(51) Alshoukr, F.; Rosant, C.; Maes, V.; Abdelhak, J.; Raguin, O.;
Burg, S.; Sarda, L.; Barbet, J.; Tourwé, D.; Pelaprat, D.; Gruaz-Guyon,
A. Novel Neurotensin Analogues for Radioisotope Targeting to
Neurotensin Receptor-Positive Tumors. Bioconjugate Chem. 2009, 20,
1602−1610.
(52) Alshoukr, F.; Prignon, A.; Brans, L.; Jallane, A.; Mendes, S.;
Talbot, J.-N.; Tourwé, D.; Barbet, J.; Gruaz-Guyon, A. Novel DOTA-
Neurotensin Analogues for 111In Scintigraphy and 68Ga PET Imaging
of Neurotensin Receptor-Positive Tumors. Bioconjugate Chem. 2011,
22, 1374−1385.
(53) Hosseinimehr, S. J.; Tolmachev, V.; Orlova, A. Liver Uptake of
Radiolabeled Targeting Proteins and Peptides: Considerations for
Targeting Peptide Conjugate Design. Drug Discovery Today 2012, 17,
1224−1232.
(54) Hodolic, M.; Wu, W. Y.; Zhao, Z.; Yu, F.; Virgolini, I.; Wang, F.
Safety and Tolerability of 68Ga-NT-20.3, a Radiopharmaceutical for
Targeting Neurotensin Receptors, in Patients with Pancreatic Ductal
Adenocarcinoma: The First in-Human Use. Eur. J. Nucl. Med. 2021,
1229.
(55) Buchegger, F.; Bonvin, F.; Kosinski, M.; Schaffland, A. O.; Prior,
J.; Reubi, J. C.; Bläuenstein, P.; Tourwé, D.; García Garayoa, E.; Bischof
8578
https://doi.org/10.1021/acs.jmedchem.1c00523
J. Med. Chem. 2021, 64, 8564−8578