Synthesis and evaluation of a 18F-labeled diarylpyrazole glycoconjugate for the imaging of NTS1-positive tumors

Article abstract of DOI:10.1021/jm401491e

Aiming to image NTS1 overexpressing tumors, the diarylpyrazole glycoconjugate 8, derived from the potent NTS1 antagonist SR142948A, was synthesized taking advantage of the palladium-catalyzed aminocarbonylation reaction. The glycoconjugate 8 displayed excellent affinity and selectivity toward NTS1. Radiosynthesis proceeded straightforwardly, obtaining [ ¹⁸F]8 with excellent stability and highly beneficial biodistribution in vivo as demonstrated by PET imaging in HT29 tumor-bearing nude mice. Thus, the tracer [¹⁸F]8 represents a highly promising candidate for PET imaging of NTS1-positive tumors.

Full text of DOI:10.1021/jm401491e

Brief Article
pubs.acs.org/jmc
18
Synthesis and Evaluation of a F‑Labeled Diarylpyrazole
Glycoconjugate for the Imaging of NTS1-Positive Tumors
Christopher Lang,† Simone Maschauer,‡ Harald Hubner,† Peter Gmeiner,† and Olaf Prante*,‡
̈
†Department of Chemistry and Pharmacy, Medicinal Chemistry, Emil Fischer Center, Friedrich Alexander University, Schuhstraße 19,
91052 Erlangen, Germany
‡Laboratory of Molecular Imaging and Radiochemistry, Department of Nuclear Medicine, Friedrich Alexander University,
Schwabachanlage 6, 91054 Erlangen, Germany
S
* Supporting Information
ABSTRACT: Aiming to image NTS1 overexpressing tumors, the diarylpyrazole glycoconjugate 8, derived from the potent
NTS1 antagonist SR142948A, was synthesized taking advantage of the palladium-catalyzed aminocarbonylation reaction. The
glycoconjugate 8 displayed excellent affinity and selectivity toward NTS1. Radiosynthesis proceeded straightforwardly, obtaining
[18F]8 with excellent stability and highly beneficial biodistribution in vivo as demonstrated by PET imaging in HT29 tumor-
bearing nude mice. Thus, the tracer [18F]8 represents a highly promising candidate for PET imaging of NTS1-positive tumors.
INTRODUCTION
■
Cancer is one of the leading causes of death worldwide. Among
the different types, lung and bronchus, prostate, colorectum,
pancreas, and breast carcinoma are especially responsible for
the high mortality.1 Therefore, the development of highly
selective agents for therapy and also diagnostics is of particular
importance. The G-protein-coupled neurotensin receptor 1
(NTS1)2,3 represents a very promising target for such agents, as
it is overexpressed in a variety of tumors including the above-
mentioned tumor types.4−6 In addition, NTS1 shows negligible
Figure 1. Structures of the peptidic NTS1 tracer 1 and the
diarylpyrazole based NTS1 antagonist 2.
expression in healthy tissues where these tumors arise from,
making it a very specific molecular target for imaging and
targeted cancer therapy.5 Until now, therapeutic approaches to
suitable for specific imaging of NTS1-positive HT29 tumors in
treat NTS1-overexpressing tumors by addressing this receptor
failed.7 However, as the abundance of NTS1 in tumors is very
nude mice by PET. However, in biodistribution studies the
tracer revealed high kidney uptake and slow renal clearance. To
specific, various efforts in the development of both diagnostic
improve this biodistribution pattern, we aimed at the
radioligands to detect NTS1-positive tumors and agents for
radiotherapy have been made.7−11 Positron emission tomog-
development of a novel nonpeptidic radioligand based on the
heterocyclic lead 2 (SR142948A, Figure 1), which has been
raphy (PET) represents in principle a highly powerful
diagnostic imaging tool, as it allows noninvasive in vivo
described as a high affinity NTS1 antagonist displaying Ki in the
low nanomolar range.15
imaging with excellent spatial resolution, high sensitivity, and
precise quantification.12 Until now, the development of NTS1
In this work, we report the synthesis of a radiotracer with
high affinity and excellent selectivity for NTS1 by taking
radiotracers that are applicable for PET was almost exclusively
advantage of our click chemistry based ligation of a terminal
based on NT(8−13), which represents the active fragment of
alkyne with 2-deoxy-2-[18F]fluoroglucosylazide. The alkyne
the peptidic endogenous NTS1 agonist neurotensin (NT, pE-
L-Y-E-N-K-P-R-R-P-Y-I-L).7,13 However, peptidic radiotracers
bearing linker unit was introduced in one step, utilizing
microwave assisted palladium-catalyzed aminocarbonylation
frequently reveal major disadvantages, primarily due to their
reaction.16−18
instability in vivo because of degradation by endogenous
peptidases,14 high kidney uptake in vivo that limits their
RESULTS AND DISCUSSION
To evaluate NTS1 binding affinity and selectivity over the
subtype NTS2, the reference compound 8 bearing the stable
applicability for radiotherapy,11 and relatively low tumor
■
retention that leads to decreased signal-to-noise ratios at late
time points after tracer injection.
fluorine isotope was synthesized according to Scheme 1. As
preliminary experiments revealed that amines bearing terminal
Recently, our group reported the synthesis of a metabolically
stable 18F-labeled NT(8−13) derivative by a click chemistry
based ligation of the terminal alkyne bearing peptide with 2-
deoxy-2-[18F]fluoroglucosylazide.10 The peptide-derived radio-
tracer 1 (Figure 1) showed excellent in vivo stability and was
Received: September 25, 2013
© XXXX American Chemical Society
A
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Journal of Medicinal Chemistry
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a
transfected in human embryonic kidney (HEK 293) cells. As
shown in Table 1, the glycoconjugate 8 showed high NTS1
Scheme 1
Table 1. Receptor Binding Data of 8 in Comparison to the
Reference Agents Neurotensin, NT(8-13) and SR142948A
a
Employing Human NTS1 and NTS2
Ki SEM (nM)
b
Ki (nM) NTS1 [3H]
c
compd
neurotensin
Ki (nM) NTS2 [3H]NT(8−13)
d
neurotensin
0.51 0.060
0.29 0.032
1.0 0.13
4.9 0.32
d
NT(8−13)
SR142948A
(2)
1.4 0.11
74 14
8
0.98 0.46
69 20
a
Ki SEM are the means of 3−10 individual experiments each done
b
in triplicate. Membranes from CHO cells stably expressing human
NTS1. Homogenates from HEK cells transiently expressing human
NTS2. Kd
experiments each done in quadruplicate.
a
Reagents and conditions: (i) (1) n-BuLi, THF, −78 °C, 1 h, (2)
c
TIPS-Cl, −78 °C, 24 h (88%); (ii) MeNH(CH2)3NHMe, KI, K2CO3,
MeCN, reflux, 69 h (89%); (iii) Mo(CO)6, Herrmann’s palladacycle,
[(t-Bu)3PH]BF4, 5, DBU, 1,4-dioxane, 140 °C, 30 min microwave
(50%); (iv) TBAF, THF, rt, 3 h (83%); (v) 2-deoxy-2-fluoroglucosyl
azide, CuSO4·5 H2O, sodium ascorbate, t-BuOH, H2O, rt, 48 h (54%).
d
SEM are the mean of 13−20 individual saturation
affinity (0.98 nM) and substantial selectivity over the subtype
NTS2 (70-fold). When compared to the affinity data of lead
structure 2 (SR142948A), the conjugated sugar moiety did not
infer the binding properties for NTS1 and NTS2 at all.
Because the glycoconjugate 8 revealed excellent in vitro
binding data, we proceeded with the radiosynthesis of 18F-
labeled 8 and evaluated the radioligand in vitro and in vivo
using tumor-bearing nude mice with NTS1-positive HT29
tumors. As shown in Scheme 2, the radiosynthesis started with
the triflate precursor 9,21 which underwent a cryptate-mediated
nucleophilic substitution with no-carrier-added [18F]fluoride.21
The acetyl protective groups of the corresponding glucose
derivative [18F]10 were subsequently hydrolyzed under basic
conditions to give unprotected [18F]11, which was employed in
the CuAAC with the building block 7 (200 nmol), affording the
NTS1 radiotracer [18F]8. The radiosynthesis followed our
recent protocol,10 but THF had to be used as a solvent for the
click conjugation step to preserve an optimal radiochemical
yield (RCY). The preparation proceeded in a total synthesis
time of 70 min, and [18F]8 was obtained in a RCY of 20 3%
(uncorrected for decay, n = 6) and a high specific activity of
35−74 GBq/μmol. The log D7.4 determination of [18F]8
revealed a value of −0.24 0.02. Stability experiments in vitro
(human serum) and in vivo displayed excellent stability of [18F]
8, as no degradation products could be observed (Supporting
Information). To demonstrate specific binding of [18F]8 in
tissue, in vitro autoradiography studies of rat brain slices were
conducted (Figure 2). The brain slices were incubated with 10
MBq of [18F]8 in the presence or absence of various
competitors.
alkynes could not be converted to the corresponding amides by
aminocarbonylation reaction, we aimed at the introduction of
an alkyne protecting group. Therefore, commercially available
6-chloro-1-hexyne was deprotonated by n-BuLi and subse-
quently treated with TIPS-Cl to give the corresponding
protected alkyne 3.19 Treatment of 3 with N,N′-dimethylpro-
pane-1,3-diamine afforded the protected diaminoalkyne 4,
which was converted to the corresponding amide 6, employing
microwave assisted palladium-catalyzed aminocarbonylation
reaction of the previously reported aryl bromide 5.20 Removal
of the protective group by TBAF led to the free alkyne 7 that
served as precursor for the radiosynthesis of [18F]8 (Scheme 2)
or underwent CuAAC with 2-deoxy-2-fluoroglucosyl azide21 to
give reference compound 8.
Receptor binding data were determined in a competition
binding assay using the radioligand [3H]neurotensin and stably
transfected Chinese hamster ovary (CHO) cells expressing
human NTS1 receptor. [3H]NT(8−13) was used for binding
assays investigating human NTS2, which was transiently
a
Scheme 2
Specific binding of [18F]8 in NTS1-positive brain areas was
demonstrated by competitive blocking with an excess of
NT(8−13) and 2 (SR142948A). The use of NT led only to
a partial decrease of radioligand binding which could be
ascribed to the presence of distinct agonist NT and antagonist
binding domains on the rat NTS1,22 in accordance with what
has been reported for various other neuropeptide receptors.23
The specific binding of [18F]8 was evident especially in brain
areas known to have a high NTS1 expression. The regional
brain distribution of [18F]8 is mainly in accordance with that of
iodine-125-labeled NT.24 Furthermore, using the NTS2
selective NT(8−13) derived peptide H-Arg-Arg-Pro-Nhtyr-
a
Reagents and conditions: (i) [18F]F−, Kryptofix 2.2.2, K2CO3,
KH2PO4, MeCN, 85 °C, 2 min (74% RCY); (ii) NaOH, 60 °C, 5
min (quant. RCY); (iii) HCl, 7, sodium ascorbate, CuSO4·5 H2O,
THF, EtOH, 60 °C, 10 min (70% RCY).
B
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demonstrated excellent tumor retention with an uptake of 0.74
0.14% ID/g at 60 min pi (Figure 3A). Consequently, the
tumor/blood ratios rapidly increased from 0.3 to 4.4 (10−60
min pi). A similar situation was observed for all other tumor/
organ ratios as depicted in Figure 3B, suggesting a suitable
signal-to-noise ratio for PET imaging. In comparison with the
HT29 tumor uptake of already described NTS1-targeting
radioligands, such as 99mTc-labeled peptides developed by
Garcia-Garayoa et al. (6% ID/g),8 68Ga- and 111In-labeled NT
derivatives by Alshoukr et al. (0.8−5% ID/g),11 and our
previously described 68Ga-labeled peptoid (0.7% ID/g)9 and
18F-glycopeptoid 1 (1.2% ID/g),10 the uptake value of [18F]8
(0.74% ID/g) is within the low range. In this context,
mutimeric ligands consisting of multiple monomeric NTS1
ligands, as recently been studied,27 could in principle lead to
increased tumor uptake; however, a suitable multimeric NTS1
PET ligand has not yet been reported. Most importantly, [18F]8
revealed an outstanding tumor/kidney ratio of 8 at 60 min pi
(Figure 3B), which is highly favorable for PET imaging and
provides the major advantage of the non-peptide [18F]8
compared with most peptide ligands that suffer from high
kidney uptake and low tumor/kidney ratios of 28,10 or even
below 1.11 When [18F]8 was injected into HT29 xenografted
nude mice for PET imaging studies, the tracer revealed
displaceable and specific NTS1-dependent tumor uptake that
was verified by co-injection of the selective NTS1 ligand
SR142948A (3 mg/kg) (Figure 4). The comparison between
Figure 2. Autoradiography of rat brain slices after incubation with
[18F]9 in the absence (control) and presence of competitors. BL,
basolateral amygdaloid nucleus; BM, basomedial amygdaloid nucleus;
Cg1, Cg2, cingulate cortex area 1/2; CPu, caudate putamen; DEn,
dorsal endopiriform nucleus; DP, dorsal pedunucular cortex; Hb,
Habenula; IL, infralimbic cortex; LSI, lateral septal nucleus; LV, lateral
ventricle; PAG, periaqueductal gray; Pir, piriform cortex; PRh,
perirhinal cortex; PrL, prelimbic cortex; RSC, retrosplenial cortex;
SN, substantia nigra; ZI, zona incerta.
Ile-Leu-OH25 (12) as competitor provided evidence for the
high NTS1 selectivity of [18F]8, as the binding of [18F]8 in
brain areas that are known to express NTS1 was fully
unaffected in the presence of 12, while binding of [18F]8 in
the marginally labeled NTS2-positive brain areas, such as
cortex, was completely diminished. Furthermore, previously
published results of autoradiography studies using a tritiated
NTS1 ligand are in high agreement with the herein obtained
results.26
As the lead structure 2 has been described to cross the
blood−brain barrier,15 we were intrigued by the question of
whether [18F]8 could enter the brain in vivo. Therefore, we
performed brain autoradiography studies with rats that were
injected with [18F]8, demonstrating that the radiotracer did not
significantly cross the blood−brain barrier (data not shown).
Using HT29 tumor-bearing nude mice with a tumor
diameter of 7−10 mm, we studied the suitability of [18F]8
for imaging NTS1 expression in peripheral tumors. As shown in
Figure 3A, the biodistribution of [18F]8 in HT29 xenografted
Figure 4. Small-animal PET imaging of [18F]8. (A) Small-animal PET
images (transaxial and coronal projections) of HT29 tumor-bearing
nude mice at 45−65 min pi of [18F]8 with (right) and without (left)
co-injection of 2 (3 mg/kg). Red arrows indicate the tumor. (B)
Tumor uptake (% ID/g) of [18F]8 in HT29 xenografted nude mice
45−65 min pi determined by μPET. Data are expressed as mean values
standard error of the mean (SEM) from 4 animals.
Figure 3. Biodistribution (A) and tumor-to-organ ratios (B) of [18F]8
in nude mice bearing HT29 xenografts. Data are expressed as mean
values SD from two animals.
control and co-injected animals demonstrated a significantly
decreased tumor uptake in the coinjected animals by 40%
(Figure 4B), thereby confirming specific visualization of NTS1
expression in HT29 tumors by [18F]8 in vivo by PET.
nude mice at 10, 30, and 60 min pi revealed fast blood
clearance, fast clearance from the liver, and negligible uptake in
the kidney, which is a highly beneficial in comparison to the
high kidney uptake of most radiopeptides. In addition, [18F]8
demonstrated high metabolic stability in vivo as determined by
radio-HPLC of blood samples taken at 10 and 30 min
postinjection (>98%). Moreover, the uptake of [18F]8 in HT29
tumors was 0.84 0.11% ID/g early at 10 min pi and the tracer
In conclusion, we successfully developed the glycoconjugate
[18F]8 as the first non-peptide tracer for in vivo PET imaging of
NTS1 expressing tumors, such as pancreatic, prostate, and
mammary carcinoma. Because of its excellent clearance
properties in vivo and high tumor retention, this tracer is a
highly promising candidate for the translation into the clinic.
Further preclinical studies of [18F]8 and derivatives thereof
using additional animal models are currently underway.
C
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Journal of Medicinal Chemistry
Brief Article
EXPERIMENTAL SECTION
REFERENCES
■
■
General. The purity of the biologically evaluated 8 was ≥95% as
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Preparation of 8. To a solution of 7 (13.5 mg, 0.02 mmol)) and
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ASSOCIATED CONTENT
■
S
* Supporting Information
1
Further experimental details, analytical data, H NMR spectra,
production of [18F]fluoride, synthesis, analysis, and evaluation
of [18F]8 (stability, log D7.4, in vivo, in vitro experiments),
binding experiments. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +49 9131 85-44440. Fax: +49 9131 85-34440. E-mail:
olaf.prante@uk-erlangen.de.
Notes
The authors declare no competing financial interest.
Schubiger, P. A. In vitro and in vivo evaluation of new radiolabeled
neurotensin(8-13) analogues with high affinity for NT1 receptors.
Nucl. Med. Biol. 2001, 28, 75−84.
ACKNOWLEDGMENTS
■
The authors thank Bianca Weigel and Manuel Geisthoff for
expert technical support. This work was supported by the
Deutsche Forschungsgemeinschaft (DFG, Grant MA 4295/1-
2).
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ABBREVIATIONS USED
■
CuAAC, copper-catalyzed azide−alkyne cycloaddition; NT,
neurotensin; NTS1, neurotensin receptor subtype 1; NTS2,
neurotensin receptor subtype 2; Nhtyr, N-2-(4-
hydroxyphenyl)ethylglycine; NLys, N-carboxymethyllysine;
RCY, radiochemical yield; Tle, tert-leucine; TIPS, triisopro-
pylsilyl
D
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