Synthesis, radiofluorination, and in vivo evaluation of novel fluorinated and iodinated radiotracers for PET imaging and targeted radionuclide therapy of melanoma

Article abstract of DOI:10.1021/jm400877v

Our project deals with a multimodal approach using a single fluorinated and iodinated melanin-targeting structure and offering both imaging (positron emission tomography (PET)/fluorine-18) and treatment (targeted radionuclide therapy/iodine-131) of melano

Full text of DOI:10.1021/jm400877v

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Article
Synthesis, radiofluorination and in vivo evaluation of
novel fluorinated and iodinated radiotracers for PET
imaging and targeted radionuclide therapy of melanoma.
Emilie M.F. Billaud, Latifa Rbah-Vidal, Aurélien Vidal, Sophie Besse, Sébastien
Tarrit, Serge Askienazy, Aurélie Maisonial, Nicole Moins, Jean-Claude
Madelmont, Elisabeth Miot-Noirault, Jean-Michel Chezal, and Philippe Auzeloux
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm400877v • Publication Date (Web): 17 Sep 2013
Downloaded from http://pubs.acs.org on September 25, 2013
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Journal of Medicinal Chemistry
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Synthesis, radiofluorination and in vivo evaluation of
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novel fluorinated and iodinated radiotracers for PET
imaging and targeted radionuclide therapy of melanoma.
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Emilie M.F. Billauda, Latifa Rbah-Vidala, Aurélien Vidala, Sophie Bessea, Sébastien Tarrita, Serge
Askienazyb, Aurélie Maisoniala, Nicole Moinsa, Jean-Claude Madelmonta, Elisabeth Miot-Noiraulta,
Jean-Michel Chezala and Philippe Auzelouxa*.
a Clermont Université, Université d'Auvergne, Imagerie Moléculaire et Thérapie Vectorisée, BP 10448,
Fꢀ63000 ClermontꢀFerrand, France. Inserm, U 990, Fꢀ63000 ClermontꢀFerrand, France.
b Cyclopharma Laboratories, Biopôle ClermontꢀLimagne, SaintꢀBeauzire, Fꢀ63360, France.
AUTHOR EMAIL ADDRESS. philippe.auzeloux@inserm.fr
RECEIVED DATE
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ABSTRACT.
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Our project deals with a multimodal approach using a single fluorinated and iodinated melaninꢀtargeting
structure and offering both imaging (Positron Emission Tomography (PET)/Fluorineꢀ18) and treatment
(targeted radionuclide therapy/Iodineꢀ131) of melanoma. Six 6ꢀiodoquinoxalineꢀ2ꢀcarboxamide
derivatives with various side chains bearing fluorine were synthesized, radiofluorinated, and their in
vivo biodistribution was studied by PET imaging in B16Bl6 primary melanomaꢀbearing mice. Among
this series, [18F]8 emerged as the most promising compound. [18F]8 was obtained by a fully automated
radiosynthesis process within 57 min with an overall radiochemical yield of 21% decayꢀcorrected. PET
imaging of [18F]8 demonstrated very encouraging results as early as 1 hour postꢀinjection with high
tumor uptake (14.33±2.11% ID/g), high contrast (11.04±2.87 tumorꢀtoꢀmuscle ratio), and favorable
clearance properties. These results, associated with the previously reported pharmacokinetic properties
and dosimetry of 8, make it a potential agent for both PET imaging and targeted radionuclide therapy of
melanoma.
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KEYWORDS. Iodinated and fluorinated radiotracers; Fluorineꢀ18; PET imaging; Biodistribution study;
Melanoma uptake
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INTRODUCTION
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Malignant melanoma affects more than 150 000 new patients per year worldwide. It accounts for up
to 90% of all deaths caused by skin cancer and displays a high metastatic potential.1 Furthermore, this
disease is the second most common cancer among patients aged 20ꢀ39.2,3
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Early melanomas (clinical stages I and II, according to the recommendations of the American Joint
Commission on Cancer) are usually treated with surgical removal of the tumor. Unfortunately, patients
with highly disseminated melanomas (stages III and IV) have a very poor prognosis, with a median
survival time of only 6ꢀ9 months and a 3ꢀyears survival rate of only 10ꢀ15%.4 These data indicate an
urgent need to find efficient therapies to treat disseminated melanoma.
Standard clinical practice for the initial treatment of cutaneous melanoma involves wide local excision
of the primary lesion and assessment of metastatic spread to lymph nodes draining the tumor site,
usually by Sentinel Lymph Node Biopsy (SLNB).5,6 Regarding malignant melanoma treatments,
chemotherapy is mostly ineffective. Monochemotherapy with dacarbazine currently represents the most
efficient treatment but the obtained response rate remains low (less than 20%) and contributes little to
overall patient survival.7,8 Despite a higher response rate of up to 40% obtained with combination of
chemotherapeutics or with combination of chemotherapy and cytokines such as interferon or
interleukinꢀ2, no significant impact on survival has been made in the past 40 years.7,9,10,11 Monoclonal
antibodies antiꢀCTLA4 (Ipilimumab) and mutated BRAFꢀV600E inhibitor (Vemurafenib) are two new
therapeutic strategies that have been tested with significant improvement of survival. However, these
two treatments induced important side effects including autoꢀimmune response (antiꢀCTLA4) and
resistance after initial antiꢀtumor response (mutated BRAFꢀV600E inhibitor).12,13
The patient’s best opportunity for a cure still remains early diagnosis of disease together with an
accurate assessment of its metastases. Imaging modalities used for malignant melanoma staging are CT,
ultrasound, MRI, and PET imaging.14 Currently, clinical data on PET/CT are solely based on
[18F]fluorodeoxyglucose ([18F]FDG) imaging, but its sensitivity is recognized to be limited for sentinel
lymph nodes detection. In these cases SLNB, typically performed following intraoperative lymphatic
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mapping, is still the gold standard.15,16,17,18 Combination of [18F]FDG PET imaging and SLNB for
evaluation of metastasis can delay definitive management planning and increase the cost of the
diagnosis process.6 [18F]FDG PET/CT also fails to highlight micrometastatic lesions that are less than
1 cm in diameter and those located mainly in lungs, liver or brain.19 Moreover, the use of this nonꢀ
specific radiotracer may be limited by the risk of falseꢀpositives due to abnormal inflammatory areas
uptakes for example. Thus, several scintigraphic studies have been developed over the past years, based
on tracers related to specific characteristics of melanoma cells.20
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Since α–MSH (αꢀmelanocyteꢀstimulating hormone) receptor MC1R (melanocortin type 1 receptor) is
overexpressed in most murine and human melanomas, it has been investigated as a target for selective
imaging and therapeutic agents.21,22,23 [18F]Labeled small synthetic peptide [18F]FBꢀNAPamide
demonstrated that it could differentiate B16/F10 and A375M with high and low MC1R expression
respectively, but this tracer showed only moderate tumor uptake and retention.24 Rheniumꢀcyclized α–
MSH analogs have also been studied in vivo ([18F]FBꢀRMSHꢀ1 and [18F]FPꢀRMSHꢀ1) displaying
specific, durable and high uptake in MC1Rꢀoverexpressing melanoma models.25,26
Different studies demonstrated that melanin pigment, detected in more than 90% of primary
melanoma cases, could be a potential target for development of imaging and therapy of melanoma.27,28,29
Recently, [18F]Nꢀ[2ꢀ(diethylamino)ethyl]ꢀ6ꢀfluoroꢀpyridineꢀ3ꢀcarboxamide ([18F]ICF01006, see Figure
1) exhibited excellent preclinical results as PET tracer for early detection of melanoma lesions in both
primary and lung colonies melanoma murine models.30,31,32 Moreover, direct comparison with [18F]FDG
showed that [18F]ICF01006 was superior in terms of contrast and specificity.32 This class of
arylcarboxamide compounds with high affinity for melaninꢀcontaining cells could also be labeled with
high energy radioisotopes (iodineꢀ131 for example) in order to induce an antiꢀtumoral effect. In
previous preclinical studies, a new iodinated quinoxalineꢀcarboxamide derivative ([131I]ICF01012,
[131I]2, see Figure 1), demonstrated a promising efficacy in a targeted radionuclide therapy (TRT)
protocol using murine and human primary melanomaꢀbearing mice models, with a tumoral
concentration primarily correlated to melanin content.33,34 Affinity studies of aromatic carboxamide
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compounds for synthetic melanin revealed the presence of two classes of binding sites, one ionic and the
other hydrophobic.28 Secondary ion mass spectrometry (SIMS) analyses also demonstrated a perfect
colocalization of ICF01012 with melanosome, illustrating its melaninꢀspecific binding.33
These highly favorable results led us to develop a new multimodal approach, designing iodinated and
fluorinated analogs of our lead radiotracer 2, suitable for both PET imaging (18Fꢀradiolabeling) and TRT
(131Iꢀradiolabeling) of melanoma.35,36 Radiofluorinated molecules were designed to select patients with
pigmented melanoma lesions, who may be included in the radionuclide therapy protocol, and to monitor
the treatment response.
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Such multimodal concept, with molecules having high affinity for melanin pigment, was first
validated with the radiotracer 3 (see Figure 1).37 However the radiolabeling of 3 with 18F was not
transposable to clinical studies due to a twoꢀpot threeꢀstep procedure associated with very low
radiochemical yields.
With the aim of discovering an appropriate clinical candidate for PET imaging and TRT of
melanoma, we designed and synthesized six iodinated and fluorinated analogs of the lead radiotracer
2.38 All derivatives had in common a 6ꢀiodoquinoxalineꢀ2ꢀcarboxamide scaffold and the side chain
bearing the fluorine atom on the tertiary amine differed, leading to saturated ethyl (4) and propyl (5)
compounds, alkene (6), alkyne (7), or pegylated structures (8 and 9) (see Figure 2). After radiolabeling
with 125I, these radiotracers were evaluated in vivo in B16 melanomaꢀbearing mice by γꢀscintigraphic
imaging: tumoral uptakes were visualized as early as 1 h post injection (p.i.) and up to 10 d p.i., in
association with high tumorꢀtoꢀmuscle ratios and a fast clearance of radioactivity from nonꢀtarget
organs. The doses which could be delivered to melanoma tumors ranged from 58.9 to
164.8 cGy/injected MBq. These very favorable dosimetry parameters are suitable for efficient targeted
radionuclide therapy of disseminated melanoma.38 Based on these promising results, herein we present
(i) the syntheses of precursors for 18Fꢀradiolabeling, (ii) fully automated radiosyntheses with 18F and (iii)
PET imaging of each tracer with tumor uptake evaluation in B16Bl6 melanomaꢀbearing mice, compared
to ex vivo biodistribution study.
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RESULTS AND DISCUSSION
Chemistry
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Radiofluorinations of compounds 5ꢀ9 required the syntheses of their corresponding mesylate
precursors (Scheme 1). The first three steps of alkyne and alkene mesylate precursors syntheses (22 and
25 respectively) were common: commercially available 2ꢀbutyneꢀ1,4ꢀdiol (10) was monoprotected
using tertꢀbutyldimethylsilyl chloride (TBDMSCl) according to a slightly modified protocol developed
by Cai et al.39 Iodination of the nonꢀprotected alcohol function of 11, in the presence of
triphenylphosphine and imidazole, afforded derivative 12. Subsequently, nucleophilic substitution using
phthalimide 1340 and potassium carbonate provided 14 in 41% yield over three steps. This key
intermediate was next used in two different synthetic pathways. First, primary amine deprotection using
hydrazine monohydrate immediately followed by peptidic coupling with activated ester 1941 provided
silylated derivative 20. After deprotection of the alcohol function with tetrabutylammonium fluoride
(TBAF), the resulting intermediate 21 was converted into the mesylate precursor 22, using
methanesulfonyl chloride (MsCl), distilled triethylamine as organic base, and catalytic 4ꢀ(N,Nꢀ
dimethylamino)pyridine (DMAP) (60 min at rt, 78% yield). Finally, compound 22 was obtained with
44% overall yield from key intermediate 14. Second, the key alkyne 14 was chemoꢀ and stereoꢀ
selectively reduced to the corresponding (E)ꢀalkene 17, in a twoꢀstep protocol adapted from Trost et
al.42 and Fürstner et al.43 Briefly, compound 14 was hydrosilylated with triethoxysilane in the presence
of ruthenium complex [Cp*Ru(MeCN)3]PF6 (1.0 mol%) to afford the crude vinylsiloxane intermediate
16. This hydrosilylation was followed by a protodesilylation under mild conditions with silver(I)
fluoride to give selectively (E)ꢀalkene 17 in excellent yield (92%). It should be noted that this twoꢀstep
protocol applied on alkyne 21 did not afford the corresponding alkene 24. Compound 17 was then
subjected to similar protocols than intermediate 14, i.e. deprotection using hydrazine monohydrate
immediately followed by peptidic coupling reaction with activated ester 19 to provide silylated
derivative 23. Finally, the deprotection of the alcohol function with TBAF was followed by the
conversion of the resulting intermediate 24 into the corresponding mesylate 25, using MsCl, distilled
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triethylamine, and DMAP (30 min at rt, 70% yield, see Scheme 2). Compound 25 was obtained with
60% overall yield from key intermediate 14.
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Using the same protocol (i.e. MsCl, distilled triethylamine and DMAP), mesylate precursors 28, 29
and 31 (see Scheme 2), bearing pegylated (n=3 or n=7) or propyl moiety were obtained in one step in a
rapid way (60ꢀ120 min) with excellent yields (92ꢀ95%) from the previously synthesized alcohols 26, 27
and 30,38 respectively.
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It should be noted that due to their limited stability, mesylate precursors 22, 25 and 31 have to be
freshly prepared and purified just before radiolabeling with 18F.
Radiochemistry
Fully automated radiosyntheses of [18F]4ꢀ9 were performed on a SynChrom R&D module (Raytest).
For all radiosyntheses, [18F]Fꢀ (Cyclopharma Laboratories) was converted into the dry [18F]KF,K222
complex. Scheme 3 describes radiosyntheses of [18F]4-9 and Table 1 summarizes conditions and results
of all radiochemistry processes.
For radiosynthesis of [18F]4, it was not possible to isolate its sulfonate precursor (data not shown) so a
oneꢀpot twoꢀstep procedure involving available stable precursors was chosen. The intermediate [18F]2ꢀ
fluoroethyl tosylate 33 was obtained by nucleophilic substitution of 1,2ꢀbis(tosyloxy)ethane 32 in
MeCN.44,45 Then, intermediate 33 immediately reacted with the secondary amine function of
quinoxaline precursor 34.41 Best radiochemical yield was obtained with a twoꢀstep heating procedure
(70 °C for 10 min then 110 °C for 10 min). After a semiꢀpreparative HPLC purification, [18F]4 was
manually formulated for biological evaluations: first, the HPLC solvent was evaporated under reduced
pressure, then the radiolabeled compound was taken up in a saline/EtOH (<5%) solution (formulation
yield was 97%). Following this procedure, [18F]4 was obtained with 11% overall radiochemical yield
(RCY), a radiochemical purity (RCP) higher than 99% and a 91 min total preparation time.
Regarding the radiosynthesis of [18F]5, the first assay was a similar oneꢀpot twoꢀstep procedure
starting from 1,3ꢀbis(tosyloxy)propane and quinoxaline derivative 34. In spite of using various reaction
conditions, a very low radiochemical yields was observed (<2%, data not shown). Thus an easier oneꢀ
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step procedure starting from mesylate precursor 31 was successfully carried out. The aliphatic
nucleophilic substitution was successfully achieved by heating a solution of 31 in MeCN with
anhydrous [18F]KF,K222 complex at 90 °C for 10 min (54% overall RCY, RCP higher than 99% and a
total preparation of 70 min).
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Compounds [18F]6, [18F]7, [18F]8 and [18F]9 were successfully radiolabeled using the same strategy
from their corresponding mesylate precursors, 25, 22, 28 and 29 respectively. Radiotracers [18F]5 and
[18F]9 were manually formulated for biological evaluations (yields were 91% and 93%, respectively).
For [18F]6-8 radiotracers, the formulation was fully automated: after dilution in saline of the collected
HPLC fraction, the radiolabeled compound was trapped on a C18 cartridge, eluted with EtOH then
saline (<10% EtOH in the final solution). Formulation yields were 76ꢀ90%. Compounds [18F]6ꢀ9 were
obtained with 16% to 34% overall RCY, RCP higher than 99% and a total preparation time ranging
from 57 to 65 min.
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Biological studies
In vivo PET imaging
The preclinical evaluation of [18F]4ꢀ9 in B16Bl6 primary melanomaꢀbearing mice by small animal
static PET imaging was performed 1 h post tracer injection to compare these tracers. This time point
was chosen following a preliminary PET imaging study at 30 min, 1 h, 2 h and 3 h post tracer injection.
Results showed that radiotracers were rapidly taken up in target tissues, with a maximal uptake at 1 h
p.i. which remained stable up to 3 h p.i. By contrast, activity in nonꢀtarget tissues such as muscle
displayed a fast washout as early as 1 h p.i. (unpublished results). Representative coronal images are
shown in Figure 3. For each of the six compounds, tumors were clearly visualized as early as 1 h p.i. For
compounds [18F]5, [18F]6, [18F]7, [18F]8 and [18F]9 significant radioactive signal was also observed in
eyes, corroborating the specific uptake in melaninꢀrich tissues. On the contrary, no significant
radioactive signal was observed in eyes for compound [18F]4 meaning that tumoral uptake may not be
due to specific accumulation. It should be noted that the experiments were performed on the strongly
pigmented C57BL/6J mouse model with a high uveal melanin content. Because of murine and human
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differences in ocular geometry as well as in melanin content, radioactivity uptake in the eyes may not be
an issue for clinical transfer.
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For all radiotracers, radioactivity in nonꢀtarget tissues was mainly located in the abdominal region in
accordance with the previously described clearance of this class of compounds via urinary and
hepatobiliary systems.28,37,38 Furthermore, very low radioactivity was detected in nonꢀtarget organs, such
as muscle, brain, heart, and lung, as early as 1 h p.i.
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Highest tumoral uptakes and tumorꢀtoꢀmuscle ratios were obtained for compounds [18F]5, [18F]6,
[18F]8 and [18F]9. Specific accumulation of radioactivity in B16Bl6 melanoma tumors was 10.56±1.76%
ID/g for [18F]5, 6.74±0.73% ID/g for [18F]6, 14.33±2.11% ID/g for [18F]8 and 6.63±1.64% ID/g for
[18F]9. These values are consistent with our previously published results (10.0±2.8% ID/g for [125I]5,
10.8±2.3% ID/g for [125I]6, 11.7±2.2% ID/g for [125I]8 and 6.8±1.8% ID/g for [125I]9).38 Tumorꢀtoꢀ
muscle ratio was 11.86±2.43 for [18F]5, 8.27±2.05 for [18F]6, 11.04±2.87 for [18F]8 and 6.62±1.90 for
[18F]9 (Table 2).
Defluorination and generation of free [18F]fluoride was negligible for [18F]4, [18F]7, [18F]8 and [18F]9
at 1 h p.i. as indicated by the low accumulation of radioactivity in the skeleton, whereas significant
uptake in bones was observed in PET images performed with [18F]5 and [18F]6.
Based on chemical stability of precursors, tracer accumulation in pigmented tissues and
defluorination, compounds [18F]8 and [18F]9 seemed to be the most relevant and promising radiotracers
for melanoma PET imaging. Compared to our previously described multimodal radiotracer [18F]3,37 a
higher melanoma uptake was observed with [18F]8 (12.72±4.45% ID/g vs. 8.30±1.70% ID/g, at 2 h p.i.;
p=0.038). Compared to [18F]1, which is currently one of the best melaninꢀtargeting PET radiotracers,
[18F]8 demonstrated a similar tumoral uptake in the same experimental model (14.33±2.11% ID/g vs.
11.44±2.67% ID/g for [18F]1 at 1 h p.i.; p=0.18) and a nearly 2ꢀfold greater tumorꢀtoꢀmuscle ratio
(11.04±2.87 vs. 6.40±1.73 for [18F]1 at 1 h p.i.; p=0.020).31,32
Ex vivo biodistribution
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Selected compounds [18F]8 and [18F]9 were studied by ex vivo biodistribution experiments by directꢀ
sampling tumors and tissues of interest at 1 h, 2 h and 3 h post tracer injection (Table 3). Uptakes in
pigmented tissues were similar to those obtained by PET imaging at 1 h p.i. and remained high and
stable until 3 h p.i. for both molecules (i.e. tumoral uptake at 3 h p.i. was 11.55±1.24% ID/g for [18F]8
and 6.76±1.35% ID/g for [18F]9). Furthermore, a significantly higher tumoral uptake was observed for
[18F]8 compared to [18F]9, at all time points (p=0.035, p=0.0016 and p=0.0001 at 1 h, 2 h and 3 h p.i.
respectively). Very low tracers accumulation in muscle and blood led to higher tumorꢀtoꢀmuscle and
tumorꢀtoꢀblood ratios reaching values of 14.46±3.06 (p=0.0007) and 14.43±1.75 (p=0.0005)
respectively for [18F]8 and values of 6.64±2.69 and 5.27±0.20 respectively for [18F]9 at 3 h p.i. Low
bone accumulation was observed at 1 h p.i. (1.40±0.10% ID/g for [18F]8 and 1.82±0.55% ID/g for
[18F]9). Partial in vivo defluorination of [18F]8 and [18F]9 occurred at 3 h p.i., which were not correlated
with in vitro stability in saline (RCP >99% for at least 6 h). Regarding radiotracer elimination, [18F]9
demonstrated a significantly higher accumulation in intestine compared to [18F]8 (p=0.018,
p=0.0000001 and p=0.02 at 1 h, 2 h and 3 h p.i. respectively), suggesting that despite a higher
hydrophilicity of [18F]9 (LogD = 0.95±0.02 vs 1.52±0.03 for [18F]8), renal excretion of this tracer was
not favored. Hence, [18F]8 derivative, bearing a triethylene glycol side chain, appeared more attractive
than compound [18F]9 bearing an heptaethylene glycol side chain.
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[18F]8 stability study.
In tumor and eyes (melaninꢀcontaining tissues), more than 99% of the radioactive signal corresponded
to unchanged [18F]8, at 1 h and 2 h p.i. (Table 4). On the contrary, a fast breakdown of [18F]8 was
observed in blood (15% of unchanged [18F]8 at 30 min) and liver (8% of unchanged [18F]8 at 1 h). In
urine samples, less than 1% of unchanged radiotracer was detected. These results demonstrated that
once linked to melanin in pigmented tissues, [18F]8 remained unchanged, whereas important and fast
metabolic processes occurred in nonꢀspecific tissues.
CONCLUSION
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Journal of Medicinal Chemistry
In summary, six [18F]fluorinated 6ꢀiodoquinoxalineꢀ2ꢀcarboxamide derivatives with various side
chains were synthesized and evaluated. [18F]4ꢀ9 were radiosynthesized on an automated module in good
yields, high radiochemical purity and displayed excellent chemical stability in physiological serum.
Biodistribution studies by in vivo PET imaging in B16Bl6 primary melanomaꢀbearing mice
demonstrated that four compounds out of six exhibited a high tropism for pigmented melanoma and also
high tumorꢀtoꢀbackground ratios as early as 1 h p.i. Regarding PET imaging results and in vivo stability,
the two best candidates [18F]8 and [18F]9 were then investigated in an ex vivo biodistribution study in
B16Bl6ꢀbearing mice. This study confirmed that derivative [18F]8 had the highest tumoral uptake in this
model and exhibited excellent pharmacokinetic features. It displayed lower uptake and faster clearance
of radioactivity from nonꢀtarget organs, with hepatobiliary/renal excretion pathways, resulting in higher
tumorꢀtoꢀblood and tumorꢀtoꢀmuscle ratios than for [18F]9.
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Thus, [18F]8 exhibited high tumoral uptake and favorable kinetics leading to highly contrasted images
as early as 1 h p.i. This work demonstrated that in terms of chemistry, radiochemistry and in vivo
biodistribution, radiotracer [18F]8 appears suitable for PET imaging.
Besides, as previously reported, [125I]8 showed high tumoral uptakes up to five days p.i. in the same
melanomaꢀbearing mice model (12.4±2.3% ID/g, 8.6±3.6% ID/g, 5.8±2.4% ID/g at 24 h, 72 h, 5 d p.i.
respectively), and a high calculated tumoral dosimetry (103.5 cGy/MBq for [131I]8).38 These results
make this compound a promising agent for targeted radionuclide therapy of melanoma (assay in
progress).
In conclusion, in our multimodality approach, radiotracer 8 appears as a good candidate for both
imaging (PET/Fluorineꢀ18) and treatment (TRT/Iodineꢀ131) of melanoma.
EXPERIMENTAL SECTION
Materials and General Methods
Chemistry. All reagents and solvents were purchased from the following commercial suppliers:
SigmaꢀAldrich, Acros Organics, ThermoFischer, Alfa Aesar, TCI Europe, Carlo Erba, SDS. All
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solvents were dried using common techniques.46 Unless otherwise noted, moisture sensitive reactions
were conducted under dry argon atmosphere. Thin layer chromatography (TLC) was performed on silica
gel 60 F254 plates or neutral aluminium oxide 60 F254 plates (Merck or SDS) and visualized with UV
light and/or developed with iodine, ninhydrin or potassium permanganate. Flash chromatography was
performed on silica gel 60A normal phase, 35–70 ꢁm (SDS). Florisil was purchased at Sigma Aldrich
(Fluka). Uncorrected melting points (mp) were recorded on ReichertꢀJungꢀKoffler apparatus. NMR
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spectra (200 MHz for H and 50 MHz for C) were recorded on a Bruker Avance 200 instrument; F
NMR spectra (470 MHz) were recorded on a Bruker DRX 500 apparatus using tetrafluorotoluene as
internal reference (δ: –63.7 ppm). δ were expressed in ppm. Infrared spectra (IR) were recorded on a
FTIR Nicolet Impact 410, a FT Vector 22 or a Nicolet IS10 with attenuated total reflectance (ATR)
accessory. Electron impact mode mass spectra (EIꢀMS) were obtained on a HP5890 series II
chromatograph coupled to HP5985B mass spectrometer. The analysis of samples was performed in
MeCN at a final concentration of 1 pmol.ꢁLꢀ1. Electrospray ionization mass spectra (ESIꢀMS) were
recorded on a EsquireꢀLC (Bruker Daltonics, Wissenbourg, France) spectrometer. The analysis of
samples was performed in MeCN at a final concentration of 1 pmol.ꢁLꢀ1. Microanalyses were
performed by Analytical Laboratory of the CNRS (Vernaison, France) for the elements indicated.
Radiochemistry. Noꢀcarrierꢀadded fluorineꢀ18 (halfꢀlife: 109.8 min) was produced via the [18O(p,
n)18F] nuclear reaction by irradiation of a 2.8 mL [18O]water target (>97%ꢀenriched, Bruce Technology,
Chapel Hill, USA) on a CPH14 cyclotron (14 MeV proton Beam, Cyclopharma laboratories, Saintꢀ
Beauzire, France). Typical production of [18F]fluoride at the end of bombardment for a 90 to 100 ꢁA.h
(120 min) irradiation: 185 GBq (5 Ci). Radiochemistry syntheses and semiꢀpreparatives HPLC were
performed using a monoꢀreactor Synchrom R&D synthesis module (Raytest, Straubenhardt, Germany).
Semiꢀpreparative HPLC were conducted on a Waters Symmetry SemiꢀPrep C18 column (300x7.8 mm;
porosity: 7 ꢁm) or Waters Spherisorb SemiꢀPrep SiO2 column (250x10 mm, porosity 10 ꢁm); flow rate:
3.0 mL.minꢀ1; temperature: rt; absorbance detection at λ = 254 nm. RadioꢀTLCs were run on Merck preꢀ
coated neutral aluminium oxide 60 F254 plates with a mixture of DCM and EtOH as solvents (98:2, v/v).
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Journal of Medicinal Chemistry
Detection of K222 was achieved using the standard chloroplatinate color test.47 Radioactivity
measurements were performed on a gamma counter equipped with a NaI(Tl) well type scintillation
counter (Model 1480 Wizard, Perkin Elmer Life Sciences, Boston, USA). Analytical HPLC were
performed on a PerkinElmer Series 200 equipped with a multiꢀwavelength UV Diode Array Detector
(DAD) and a GABI Star gammaꢀdetector (Raytest, Straubenhardt, Germany) at a flow rate of 0.9 or
1.0 mL.minꢀ1. Column: Waters Symmetry C18 (150x4.6 mm; porosity: 5 ꢁm) or Waters Spherisorb
SiO2 (150x4.6 mm, porosity 5 ꢁm); temperature: rt; absorbance detection at λ = 254 nm. Purity and
specific activity were determined by analytical HPLC. For each tested compound, purity was >99%.
The identity of each [18F]fluorinated compound was confirmed by coelution with its nonꢀradioactive
counterpart. A delay time of 0.35 min was observed between the first detector (UV) and the second
(gamma).
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Octanol-water distribution coefficient (LogD). Approximately 74 kBq of radiotracer was added to a
mixture of PBS (pH 7.4, 1.0 mL) and octanol (1.0 mL) in an Eppendorf microcentrifuge tube. The
mixture was vigorously vortexed for 1 min, three times at rt. After centrifugation (3,000 rpm, 5 min),
aliquots (100 ꢁL) of both layers were sampled and the radioactivity was measured using the gamma
counter. Three independent experiments were performed in duplicates.
Cell culture. The B16Bl6 syngenic melanoma cells were obtained from Dr Fidler Laboratories (Texas
University, Houston, USA). Cells cultures were maintained as monolayers in Dulbecco’s modified
Eagle’s medium (DMEM)/Glutamax(Invitrogen, Cergy Pontoise, France) supplemented with 10% calf
serum (Sigma, Saint Quentin, Fallavier, France), 1% vitamins (Invitrogen), 1 mM sodium pyruvate
(Invitrogen), 1% nonꢀessential amino acids (Invitrogen) and 4 ꢁg.mLꢀ1 of gentamycin base (Invitrogen).
The cells were grown at 37 °C in a humidified incubator containing 5% CO2.
Primary murine melanoma model. Animals were handled and cared in accordance with the guidelines
for the Care and Use of Laboratory Animals (National Research Council, 1996) and European Directive
86/609/EEC. Protocols were performed under the authorization of the French Direction des Services
Vétérinaires (authorization no. CE 86ꢀ12) and conducted under the supervision of authorized
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investigators in accordance with the institution’s recommendations for the use of laboratory animals.
C57BL/6J male mice (6ꢀ8 weeks old) were obtained from Charles River (l’Arbresle, France). Cells in
exponential growth phase were trypsinized, washed with phosphate buffer saline (PBS), and
resuspended in PBS. Mice anesthetized by isoflurane (2%) inhalation were inoculated with 3x105
melanoma B16Bl6 cells in PBS (0.1 mL) by subcutaneous injection on the right shoulder.
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In vivo PET imaging. PET imaging sessions were performed at dayꢀ10 or dayꢀ14 after inoculation
(tumoral volume range of 431.8 ± 154.3 mm3). Wholeꢀbody PET scans were acquired 1 h after
intravenous injection of [18F]labeled compound (9ꢀ12 MBq/0.15 mL) via the tail vein (range 2ꢀ8
animals/compound) and were anesthetized by intraperitoneal administration (200 ꢁL/20 gꢀmouse) of a
mixture of ketamine (Imalgene 500, Rhône Mérieux, Lyon, France) and xylazine (Rompun, Bayer,
France) in saline, 4:1 ratio. Wholeꢀbody acquisition (25 minꢀduration) was performed using a smallꢀ
animal PET device (eXplore Vista, GE Healthcare) with a 250ꢀ700 keV energy window set and 6 ns
coincidenceꢀtiming window. The spatial resolution of this system was 1.4 mm Full Width at Half
Maximum at the center of the Field Of View.48 Images were reconstructed using a 2D orderedꢀsubset
expectation maximization (Fore/2D OSEM) method including corrections for scanner dead time, scatter
radiations and randoms. No corrections were applied for partial volume or attenuation.
Regional radiotracer uptake was quantified by standard RegionꢀOfꢀInterest (ROI) analysis with
eXplore Vista software package (GE Healthcare) as previously described.32 Briefly, on decayꢀcorrected
wholeꢀbody coronal images, a manual 2DꢀROI was drawn around the whole tumor and different organs.
The reference tissue ROI was drawn on caudal thigh muscle and considered as background. The mean
counts per pixel per minute were obtained from the ROI and converted to counts per milliliter per min
using a calibration constant. By assuming a tissue density of 1 g.mLꢀ1, the ROIs were converted to
counts/g/min. An ROIꢀderived % ID/g of tissue was then determined by dividing counts per gram per
minute by injected dose. In vivo tumorꢀtoꢀbackground ratio was calculated by dividing the % ID/g value
of tumor by the % ID/g value of muscle.
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Ex vivo biodistribution studies. Ex vivo biodistribution study was performed for [18F]8 and [18F]9.
Experiments were undertaken at dayꢀ10 or dayꢀ14 after cells implantation (n=3ꢀ6 animals/group).
Animals were sacrificed by CO2 asphyxiation 1 h, 2 h and 3 after i.v. injection of [18F]labeled
compound (9ꢀ12 MBq). Tumors, major organs and tissues were promptly excised, harvested, weighted
and their radioactivity counted. After radioactive decay correction, results were expressed as % ID/g.
[18F]8 stability study. Six primary melanomaꢀbearing mice were injected with [18F]8 (18 MBq/i.v.)
and sacrificed 30 min, 1 h and 2 h p.i. To determine the metabolic stability of [18F]8, blood, liver, urine,
eyes and tumor samples were collected. Blood was immediately centrifuged for 5 min at 3,000 rpm and
plasma directly investigated by analytical HPLC. Urine sample was collected in the bladder and directly
investigated by analytical HPLC. Tumors, eyes and livers were pooled, suspended in MeOH/NH4OH
99.8:0.2 (5 mL), homogenized using GentleMACS dissociator (Miltenyi Biotec, Bergisch Gladbach,
Germany) and centrifuged for 5 min at 3,000 rpm. The extraction efficiency was determined by
measuring the radioactivity of supernatants and precipitates (extraction yields: 61%, 55% and 99%
respectively). Supernatants were passed through a 0.22 ꢁm Millipore filter and then concentrated to a
volume between 200 and 500 ꢁL before injection (duplicate) onto the analytical HPLC (C18 column;
MeOH/H2O/NH4OH 0:99.8:0.2 to 29.9:69.9:0.2 v/v/v linear gradient in 8 min then 29.9:69.9:0.2 to
59.9:39.9:0.2 linear gradient in 1 min then 59.9:39.9:0.2 to 89.9:9.9:0.2 linear gradient in 8 min then
0:99.8:0.2 isocratic during 8 min; 0.9 mL.minꢀ1).
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Statistical Analysis. Quantitative data are expressed as mean ± standard deviation (SD). Means were
compared using Student t test. Values were considered statistically significant for p<0.05.
Chemistry
N-[2-[N-Ethyl-N-(2-fluoroethyl)amino]ethyl]-6-iodoquinoxaline-2-carboxamide
4.
Fluorineꢀ19
standard compound 4 was prepared as described by Maisonial, Billaud et al.38 Rf (Al2O3, DCM/EtOH,
1
98:2, v/v) 0.72; IR (CCl4) ν 1474, 1522, 1685, 2855, 2927 cmꢀ1; H NMR (CDCl3) δ 1.12 (t, 3H, J =
3
7.1 Hz), 2.75 (q, 2H, J = 7.1 Hz), 2.87 (t, 1H, J = 6.0 Hz), 2.92 (dt, 2H, JHꢀF = 26.8 Hz, J = 5.0 Hz),
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2
3.62 (q, 2H, J = 6.0 Hz), 4.58 (dt, 2H, JHꢀF = 47.7 Hz, J = 5.0 Hz), 7.81 (d, 1H, J = 8.8 Hz), 8.05 (dd,
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1H, J = 8.8, 1.8 Hz), 8.45 (br, 1H), 8.58 (d, 1H, J = 1.8 Hz), 9.62 (s, 1H); C NMR (CDCl3) δ 11.8,
4
5
6
37.3, 48.5, 52.9, 53.6 (d, 2JCꢀF = 20 Hz), 82.5 (d, 1JCꢀF = 167 Hz), 98.0, 130.9, 138.6, 139.6, 139.7, 144.0,
144.4, 144.6, 163.1; 19F NMR (CDCl3) δ –220.22; MS m/z 416 (M+, 1), 104 (100), 76 (12), 56 (8).
7
8
9
N-[2-[N-Ethyl-N-(3-fluoropropyl)amino]ethyl]-6-iodoquinoxaline-2-carboxamide
5.
Fluorineꢀ19
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standard compound 5 was prepared as described by Maisonial, Billaud et al.38 Rf (SiO2, ethyl acetate)
0.36; 1H NMR (CD3CN) δ 1.05 (t, 3H, J = 7.1 Hz), 1.82 (m, 2H), 2.64 (m, 6H), 3.52 (q, 2H, J = 5.9 Hz),
4.56 (dt, 2H, 2JH-F = 47.4 Hz, J = 6.0 Hz),, 7.87 (d, 1H, J = 8.8 Hz), 8.08 (dd, 1H, J = 8.8, 1.9 Hz), 8.41
4
(br, 1H), 8.60 (d, 1H, J = 1.9 Hz), 9.63 (s, 1H); 13C NMR (CD3CN) δ 11.5, 28.1 (d, JCꢀF = 1 Hz), 37.3,
3
1
47.7, 52.3, 52.9 (d, JCꢀF = 11 Hz), 82.7 (d, JCꢀF = 166 Hz), 98.2, 131.0, 138.6, 139.5, 139.8, 143.8,
144.4, 144.6, 162.2; 19F NMR (CD3CN) δ –220.47; ESIꢀMS m/z [M+H]+ 431.10.
N-[2-[N-Ethyl-N-[(E)-4-fluorobut-2-enyl]amino]ethyl]-6-iodoquinoxaline-2-carboxamide
6.
Fluorineꢀ19 standard compound 6 was prepared as described by Maisonial, Billaud et al.38 Rf (Al2O3,
DCM/EtOH, 99:1, v/v) 0.36; mp 50ꢀ52 °C; IR (CCl4) ν 1160, 1353, 1475, 1520, 1682, 2817, 2850ꢀ
3000, 3409 cmꢀ1; 1H NMR (CDCl3) δ 1.08 (t, 3H, J = 7.1 Hz), 2.63 (q, 2H, J = 7.1 Hz), 2.72 (t, 2H, J =
6.2 Hz), 3.21 (m, 2H), 3.57 (q, 2H, J = 5.9 Hz), 4.81 (dd, 2H, 2JHꢀF = 47.0 Hz, J = 4.3 Hz), 5.90 (m, 2H),
7.81 (d, 1H, J = 8.8 Hz), 8.07 (dd, 1H, J = 8.8, 1.8 Hz), 8.32 (m, 1H), 8.59 (d, 1H, J = 1.8 Hz), 9.63 (s,
13
4
1
1H); C NMR (CDCl3) δ 12.2, 37.4, 47.8, 51.9, 55.2 (d, JCꢀF = 1 Hz), 83.0 (d, JCꢀF = 162 Hz), 98.1,
127.4 (d, 2JCꢀF = 17 Hz), 130.9, 133.2 (d, 3JCꢀF = 12 Hz), 138.7, 139.7, 139.8, 144.1, 144.5, 144.7, 163.0;
19F NMR (CDCl3) δ –211.13 (t, 2JHꢀF = 48.2 Hz); ESIꢀMS m/z [M+H]+ 442.90.
N-[2-[[N-Ethyl-N-(4-fluorobut-2-ynyl)]amino]ethyl]-6-iodoquinoxaline-2-carboxamide 7. Fluorineꢀ
19 standard compound 7 was prepared as described by Maisonial, Billaud et al.38 Rf (Al2O3,
DCM/EtOH, 99:1, v/v) 0.86; IR (CCl4) ν 1475, 1522, 1685, 2928 cmꢀ1; 1H NMR (acetoneꢀd6) δ 1.11 (t,
2
3H, J = 7.2 Hz), 2.64 (q, 2H, J = 7.2 Hz), 2.80 (t, 2H, J = 6.1 Hz), 3.59 (m, 4H), 4.97 (dt, 2H, JHꢀF
=
47.5 Hz, J = 1.7 Hz), 7.80 (d, 1H, J = 8.8 Hz), 8.05 (dd, 1H, J = 8.8, 1.8 Hz), 8.25 (br, 1H), 8.58 (d, 1H,
J = 1.8 Hz), 9.62 (s, 1H); 13C NMR (acetoneꢀd6) δ 12.9, 37.7, 42.2 (d, 4JCꢀF = 3 Hz), 48.3, 52.9, 71.5 (d,
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1JCꢀF = 161 Hz), 80.3 (d, 2JCꢀF = 22 Hz), 85.5 (d, 3JCꢀF = 12 Hz), 98.3, 131.8, 139.1, 140.2, 140.5, 145.0,
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145.3, 145.5, 163.5; 19F NMR (acetoneꢀd6) δ –213.79; ESIꢀMS m/z [M+H]+ 441.10.
4
5
6
N-(12-ethyl-1-fluoro-3,6,9-trioxa-12-azatetradecan-14-yl)-6-iodoquinoxaline-2-carboxamide
8.
7
8
9
Fluorineꢀ19 standard compound 8 was prepared as described by Maisonial, Billaud et al.38 Rf (SiO2,
ethyl acetate/EtOH, 85:15, v/v) 0.30; IR (ATR diamond accessory) ν 1046, 1107, 1352, 1473, 1523,
1592, 1669, 2868, 2916, 3300ꢀ3400 cmꢀ1; 1H NMR (CDCl3) δ 1.07 (t, 3H, J = 7.1 Hz), 2.65 (q, 2H, J =
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2
7.1 Hz), 2.77 (m, 4H), 3.61 (m, 12H), 3.69 (m, 2H), 4.53 (dt, 2H, JHꢀF = 47.7 Hz, J = 4.1 Hz), 7.81 (d,
1H, J = 8.8 Hz), 8.07 (dd, 1H, J = 8.8, 1.8 Hz), 8.42 (br, 1H), 8.60 (d, 1H, J = 1.8 Hz), 9.63 (s, 1H); 13C
NMR (CDCl3) δ 11.5, 37.1, 48.9, 52.8 (2C), 70.8 (6C), 83.2 (1JCꢀF = 169 Hz), 98.1, 131.0, 138.6, 139.5,
139.7, 144.1, 144.4, 144.7, 163.3; 19F NMR (CDCl3) δ –223.1; ESIꢀMS m/z [M+H]+ 549.20.
N-(24-ethyl-1-fluoro-3,6,9,12,15,18,21-heptaoxa-24-azahexacosan-26-yl)-6-iodoquinoxaline-2-
carboxamide 9. Fluorineꢀ19 standard compound 9 was prepared as described by Maisonial, Billaud et
al.38 Rf (SiO2, ethyl acetate/EtOH, 5:5, v/v containing 0.5% of NH4OH) 0.32; IR (ATR diamond
accessory) ν 1129, 1353, 1474, 1527, 1593, 1675, 2800ꢀ3000, 3300ꢀ3400 cmꢀ1; 1H NMR (CDCl3) δ 1.07
(t, 3H, J = 7.1 Hz), 2.67 (q, 2H, J = 7.1 Hz), 2.79 (m, 4H), 3.61 (m, 30H), 4.55 (dt, 2H, 2JHꢀF = 47.8 Hz,
J = 4.1 Hz), 7.83 (d, 1H, J = 8.8 Hz), 8.08 (dd, 1H, J = 8.8, 1.6 Hz), 8.46 (br, 1H), 8.61 (d, 1H, J =
1
1.6 Hz), 9.64 (s, 1H);13C NMR (CDCl3) δ 11.5, 37.0, 48.0, 52.9, 53.0, 70.6 (14C), 83.3 (d, JC-F
=
163 Hz), 98.1, 131.0, 138.6, 139.7, 139.7, 144.1, 144.4, 144.7, 163.3; 19F NMR (CDCl3) δ –223.2; ESIꢀ
MS m/z [M+H]+ 725.20.
4-(tert-butyldimethylsilyloxy)but-2-yn-1-ol (11). To a solution of 2ꢀbutyneꢀ1,4ꢀdiol 10 (10.0 g,
116 mmol) in anhydrous N,Nꢀdimethylformamide (DMF) (125 mL) were added successively, imidazole
(5.9 g, 87.1 mmol) and TBDMSCl (10.5 g, 69.7 mmol). The mixture was stirred at rt for 24 h. The
reaction was quenched with MeOH (40 mL) and water (120 mL) and then extracted with ethyl acetate
(3 x 200 mL). The organic layers were combined, washed with brine (200 mL), dried on MgSO4,
filtered and evaporated under reduced pressure. The residue was taken up in ethyl acetate (200 mL) and
washed with water (3 x 200 mL). The organic layer was dried on MgSO4, filtered and evaporated under
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reduced pressure. The crude product was purified by chromatography (SiO2, DCM/EtOH, 98:2, v/v) to
give compound 11 (9.45 g, 47.2 mmol) as a yellow oil. Yield 68%; Rf (SiO2, DCM/EtOH, 98:2, v/v)
0.35; IR (NaCl) ν 778, 837, 1084, 1256, 1472, 2350, 2859, 2930, 3200–3500 cmꢀ1. 1H NMR (CDCl3) δ
0.09 (s, 6H), 0.87 (s, 9H), 2.93 (br, 1H), 4.25 (s, 2H), 4.31 (s, 2H). 13C NMR (CDCl3) δ ꢀ5.1 (2C), 18.4,
25.9 (3C), 51.2, 51.8, 83.1, 84.4. EIꢀMS m/z 185 (<1) 143 (3), 126 (3), 125 (28), 85 (5), 77 (6), 76 (8),
75 (100), 73 (7), 61 (3), 59 (3), 57 (3).
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tert-butyl(4-iodobut-2-ynyloxy)dimethylsilane (12). To a solution of alcohol 11 (9.35 g, 46.7 mmol)
vigorously stirred at 0 °C in anhydrous DCM (250 mL) were added successively, imidazole (4.13 g,
60.7 mmol), triphenylphosphine (15.9 g, 60.7 mmol) and iodine (15.4 g, 60.7 mmol). The mixture was
stirred at 0 °C for 30 min and allowed to stand at rt for 1 h before addition of an aqueous solution of
10% sodium bisulfite (320 mL). After decanting, the aqueous layer was extracted with DCM (3 x
150 mL). The organic layers were combined, dried on MgSO4, filtered and evaporated under reduced
pressure. The residue containing the desired product was triturated in nꢀpentane (280 mL) and stirred for
45 min. The precipitate was filtered and washed with nꢀpentane (50 mL). The filtrate was evaporated
under reduced pressure and the crude product was purified by chromatography (SiO2, nꢀpentane/diethyl
ether, 99:1, v/v) to give compound 12 (11.2 g, 36.1 mmol) as a pale yellow oil. Yield 78%; Rf (SiO2, nꢀ
pentane/diethyl ether, 99:1, v/v) 0.30; IR (NaCl) ν 778, 837, 1086, 1255, 1471, 2350, 2857, 2929 cmꢀ1.
1H NMR (CDCl3) δ 0.11 (s, 6H), 0.89 (s, 9H), 3.71 (m, 2H), 4.31 (m, 2H). 13C NMR (CDCl3) δ ꢀ18.5, ꢀ
5.1 (2C), 18.4, 25.9 (3C), 51.9, 81.6, 84.0.
N-[2-[N-[4-(tert-butyldimethylsilyloxy)but-2-ynyl]-N-ethylamino]ethyl]phthalimide (14). To
a
solution of 2ꢀ[2ꢀ(ethylamino)ethyl]ꢀ1Hꢀphthalimide hydrochloride 13 (4.51 g, 17.7 mmol)40 in
anhydrous MeCN (85 mL) were successively added potassium carbonate (2.45 g, 17.7 mmol) and
compound 12 (5.50 g, 17.7 mmol). The mixture was stirred at rt for 72 h. Saturated aqueous sodium
carbonate solution (250 mL) was added, and the aqueous layer was extracted with DCM (3 x 150 mL).
The organic layers were combined, dried on MgSO4, filtered and evaporated under reduced pressure.
The obtained residue was purified by chromatography (SiO2, DCM/EtOH, 8:2, v/v) to give compound
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Journal of Medicinal Chemistry
14 (5.54 g, 13.8 mmol) as an orange solid. Yield 78%; Rf (SiO2, DCM/EtOH, 8:2, v/v) 0.85; mp 49–
1
2
3
4
1
51 °C. IR (NaCl) ν 777, 837, 1032, 1254, 1335, 1401, 1431, 1470, 1706, 2273, 2858, 2929 cmꢀ1. H
5
6
7
8
NMR (CDCl3) δ 0.10 (s, 6H), 0.88 (s, 9H), 0.96 (t, 3H, J = 7.1 Hz), 2.56 (q, 2H, J = 7.1 Hz), 2.79 (t, 2H,
J = 6.4 Hz), 3.51 (s, 2H), 3.78 (t, 2H, J = 6.4 Hz), 4.31 (t, 2H, J = 1.7 Hz), 7.70 (m, 2H), 7.80 (m, 2H).
13C NMR (CDCl3) δ ꢀ5.0 (2C), 12.8, 18.4, 25.9 (3C), 35.9, 41.8, 47.6, 50.8, 51.8, 79.4, 83.9, 123.3 (2C),
132.3 (2C), 133.9 (2C), 168.5 (2C). ESIꢀMS m/z [M + H]+ 401.26.
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13
14
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N-[4-(tert-butyldimethylsilyloxy)but-2-ynyl]-N-ethyl-1,2-ethylenediamine (15). To a solution of
phthalimide 14 (5.54 g, 13.8 mmol) in EtOH (500 mL) was added hydrazine monohydrate (6.71 mL,
138 mmol, 10 equiv). The mixture was stirred at rt for 17 h. After cooling to 0 °C for 4 h, the precipitate
was filtered and washed with EtOH (50 mL). The filtrate was evaporated under reduced pressure and
taken up in cold EtOH (50 mL), filtered and washed with cold EtOH (50 mL). Last steps were repeated
until no more white solid appeared after evaporation of the filtrate. Compound 15 (3.74 g, 13.8 mmol)
was obtained as a yellow oil and used without further purification. Yield quant.; Rf (Al2O3, DCM/EtOH,
8:2, v/v) 0.23; IR (ATR diamond accessory) ν 837, 1116, 1255, 1472, 1641, 2275, 2858, 2931 cmꢀ1. 1H
NMR (CDCl3) δ 0.11 (s, 6H), 0.90 (s, 9H), 1.05 (t, 3H, J = 7.1 Hz), 2.59 (q, 2H, J = 7.1 Hz), 2.71 (t, 2H,
13
J = 5.5 Hz), 3.23 (m, 2H), 3.44 (s, 2H), 4.32 (s, 2H). C NMR (CDCl3) δ ꢀ5.0 (2C), 12.8, 18.4, 25.9
(3C), 37.9, 41.5, 47.5, 51.8, 52.4, 79.1, 84.0.
N-[2-[N-[(E)-4-(tert-butyldimethylsilyloxy)but-2-enyl]-N-ethylamino]ethyl]phthalimide (17). This
protocol was adapted from Trost et al.42 and Fürstner et al.43 To a solution of phthalimide 14 (525 mg,
1.31 mmol) in anhydrous DCM (8 mL) were successively added at 0 °C, triethoxysilane (290 ꢁL,
1.57 mmol) and catalyst [Cp*Ru(MeCN)3]PF6 (7.0 mg, 13.1 ꢁmol). The mixture was stirred at 0 °C for
10 min and then at rt for 2 h. Diethyl ether was added (25 mL), and the mixture was filtered on a Florisil
pad (100ꢀ200 mesh). The pad was washed with diethyl ether (50 mL), DCM (25 mL), and then the
filtrate was evaporated under reduced pressure. The obtained residue (710 mg, 1.26 mmol) was taken up
in tetrahydrofuran (THF) (11 mL), and a suspension of silver fluoride 1M in MeOH (2.5 mL,
2.51 mmol) was added. The mixture was stirred at rt in darkness for 2.5 h, filtered and then washed with
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ether (20 mL) and ethyl acetate (20 mL). The filtrate was evaporated under reduced pressure. The
obtained residue was purified by chromatography (SiO2, ethyl acetate/cyclohexane, 3:7, v/v) to give
compound 17 (485 mg, 1.21 mmol) as a pale yellow oil. Yield 92%; Rf (SiO2, ethyl
acetate/cyclohexane, 3:7, v/v) 0.25; IR (ATR diamond accessory) ν 778, 839, 1101, 1257, 1396, 1469,
1
2
3
4
5
6
7
8
9
1
1718, 2856, 2930 cmꢀ1. H NMR (500 MHz, CDCl3) δ 0.07 (s, 6H), 0.89 (s, 9H), 0.97 (t, 3H, J =
10
11
12
13
14
15
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17
18
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7.1 Hz), 2.56 (q, 2H, J = 7.1 Hz), 2.71 (t, 2H, J = 6.8 Hz), 3.14 (d, 2H, J = 5.9 Hz), 3.76 (t, 2H, J =
6.8 Hz), 4.11 (d, 2H, J = 4.7 Hz), 5.61 (dt, 1H, J = 15.5, 5.9 Hz), 5.66 (dt, 1H, J = 15.5, 4.7 Hz), 7.70
(m, 2H), 7.83 (m, 2H). 13C NMR (CDCl3) δ ꢀ5.1 (2C), 12.1, 18.4, 26.0 (3C), 36.1, 47.4, 50.5, 55.3, 63.6,
123.2 (2C), 127.4, 132.3 (2C), 132.5, 133.9 (2C), 168.5 (2C). ESIꢀMS m/z [M + H]+ 403.32.
N-[(E)-4-(tert-butyldimethylsilyloxy)but-2-enyl]-N-ethyl-1,2-ethylenediamine (18). Compound 18 was
prepared following the same procedure described above for 15, starting from phthalimide 17 (250 mg,
0.62 mmol). Compound 18 (169 mg, 0.62 mmol) was obtained as a pale yellow oil and used without
1
further purification. Yield quant.; Rf (SiO2, ethyl acetate/cyclohexane, 1:1, v/v) 0.12; H NMR
(CDCl3) δ 0.05 (s, 6H), 0.90 (s, 9H), 0.99 (t, 3H, J = 7.1 Hz), 2.50 (m, 4H), 2.60 (br, 2H), 2.73 (t, 2H, J
13
= 6.1 Hz), 3.07 (m, 2H), 4.13 (m, 2H), 5.67 (m, 2H). C NMR (CDCl3) δ ꢀ5.1(2C), 11.9, 18.6, 26.0
(3C), 39.6, 47.6, 55.4, 55.8, 63.6, 127.6, 132.3.
N-[2-[[N-Ethyl-N-(4-(tert-butyldimethylsilyloxy)but-2-ynyl)]amino]ethyl]-6-iodoquinoxaline-2-
carboxamide (20). To a suspension of activated ester 4ꢀnitrophenyl 6ꢀiodoquinoxalineꢀ2ꢀcarboxylate 19
(1.00 g, 2.38 mmol) in anhydrous THF (10 mL) was added a solution of compound 15 (0.71 g,
2.61 mmol) in anhydrous THF (15 mL). The mixture was stirred at rt for 18 h. The solvent was
evaporated under reduced pressure, the residue was taken up in DCM (25 mL) and the mixture was
poured into a 1M aqueous NaOH solution (75 mL). The aqueous layer was extracted with DCM
(3 x 25 mL), the combined organic layers were washed with a 5% aqueous sodium carbonate solution
(2 x 75 mL), dried on MgSO4, filtered and evaporated under reduced pressure. The obtained residue was
purified by chromatography (SiO2, ethyl acetate/nꢀpentane, 9:1, v/v) to give compound 20 (820 mg,
1.48 mmol) as a yellow gum. Yield 63%; Rf (SiO2, ethyl acetate/nꢀpentane, 9:1, v/v) 0.42; IR (NaCl) ν
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407, 777, 836, 1074, 1253, 1353, 1387, 1472, 1472, 1518, 1593, 1656, 1683, 2273, 2858, 2929,
1
2
3
1
3363 cmꢀ1. H NMR (CDCl3) δ 0.08 (s, 6H), 0.82 (s, 9H), 1.04 (t, 3H, J = 7.1 Hz), 2.57 (q, 2H, J =
4
5
6
7
8
7.1 Hz), 2.74 (t, 2H, J = 5.9 Hz), 3.46 (br, 2H), 3.53 (q, 2H, J = 5.9 Hz), 4.27 (br, 2H), 7.72 (d, 1H, J
=8.9 Hz), 7.94 (dd, 1H, J = 8.9, 1.8 Hz), 8.22 (m, 1H), 8.47 (d, 1H, J = 1.8 Hz), 9.54 (s, 1H). 13C NMR
(CDCl3) δ ꢀ5.1 (2C), 13.0, 18.3, 25.8 (3C), 37.1, 41.7, 47.6, 51.8, 51.9, 79.4, 83.9, 98.0, 130.8, 138.5,
139.4, 139.6, 144.0, 144.3, 144.6, 162.9. ESIꢀMS m/z [M + H]+ 553.17.
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N-[2-[[N-Ethyl-N-(4-hydroxybut-2-ynyl)]amino]ethyl]-6-iodoquinoxaline-2-carboxamide (21). To a
solution of protected alcohol 20 (200 mg, 0.36 mmol) in THF (6 mL) was added a 1M solution of TBAF
in THF (543 ꢁL, 0.54 mmol). The mixture was stirred at rt for 2 h. The reaction was quenched by
addition of a saturated aqueous sodium hydrogencarbonate solution (60 mL), followed by distilled water
(30 mL) and then ethyl acetate (30 mL). The mixture was extracted with ethyl acetate (3 x 30 mL). The
organic layers were combined, washed with brine (30 mL), dried on MgSO4, filtered and evaporated
under reduced pressure. The obtained residue was purified by chromatography (SiO2, ethyl acetate) to
give compound 21 (140 mg, 0.32 mmol) as a yellow solid. Yield 89%; Rf (SiO2, ethyl acetate) 0.17; mp
119–121 °C. IR (NaCl) ν 407, 1022, 1353, 1473, 1523, 1592, 1664, 2273, 2865, 2925, 3200–3600 cmꢀ1.
1H NMR (CDCl3) δ 1.10 (t, 3H, J = 7.1 Hz), 2.62 (q, 2H, J = 7.1 Hz), 2.74 (t, 2H, J = 6.1 Hz), 3.52 (br,
2H), 3.60 (q, 2H, J = 6.1 Hz), 4.29 (t, 2H, J = 1.8 Hz), 7.81 (d, 1H, J = 8.9 Hz), 8.03 (dd, 1H, J = 8.9,
1.8 Hz), 8.29 (m, 1H), 8.57 (d, 1H, J = 1.7 Hz), 9.61 (s, 1H). 13C NMR (CDCl3) δ 12.9, 37.3, 42.0, 48.0,
51.0, 52.0, 80.2, 83.9, 98.2, 130.8, 138.6, 139.5, 139.8, 143.9, 144.4, 144.6, 163.1. ESIꢀMS m/z [M +
H]+ 439.09.
N-[2-[N-Ethyl-N-[(E)-4-(tert-butyldimethylsilyloxy)but-2-enyl)]amino]ethyl]-6-iodoquinoxaline-2-
carboxamide (23). Compound 23 was prepared following the same procedure described above for 20,
using compound 18 (147 mg, 0.54 mmol). Protected alcohol 23 (180 mg, 0.32 mmol) was obtained as a
yellow oil. Yield 93%; Rf (SiO2, ethyl acetate) 0.43; IR (ATR diamond accessory) ν 778, 838, 1102,
1
1257, 1354, 1386, 1473, 1525, 1593, 1683, 2855, 2929, 3300–3400 cmꢀ1. H NMR (CDCl3) δ 0.00 (s,
6H), 0.83 (s, 9H), 1.05 (t, 3H, J = 7.1 Hz), 2.56 (q, 2H, J = 7.1 Hz), 2.70 (t, 2H, J = 6.0 Hz), 3.16 (br,
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2H), 3.56 (q, 2H, J = 6.0 Hz), 4.11 (br, 2H), 5.72 (m, 2H), 7.79 (d, 1H, J = 8.8 Hz), 8.03 (dd, 1H, J =
1
2
3
13
8.8, 1.8 Hz), 8.35 (m, 1H), 8.57 (d, 1H, J = 1.8 Hz), 9.61 (s, 1H). C NMR (CDCl3) δ ꢀ5.2 (2C), 12.2,
4
5
6
7
8
18.4, 26.0 (3C), 37.3, 47.5, 51.6, 55.3, 63.5, 97.9, 127.3, 130.9, 132.6, 138.6, 139.6 (2C), 144.1, 144.4,
144.7, 162.9. ESIꢀMS m/z [M + H]+ 555.21.
9
N-[2-[N-Ethyl-N-[(E)-4-hydroxybut-2-enyl]amino]ethyl]-6-iodoquinoxaline-2-carboxamide
(24).
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Compound 24 was prepared following the same procedure described above for 21, starting from 23
(171 mg, 0.31 mmol). Alcohol 24 (135 mg, 0.31 mmol) was obtained as a yellow oil. Yield 99%; Rf
(SiO2, DCM/EtOH, 9:1, v/v) 0.20; IR (ATR diamond accessory) ν 978, 1355, 1475, 1533, 1593, 1675,
1
2823, 2934, 3200–3500 cmꢀ1. H NMR (500 MHz, CDCl3) δ 1.08 (t, 3H, J = 7.1 Hz), 2.63 (q, 2H, J =
7.1 Hz), 2.73 (t, 2H, J = 6.2 Hz), 3.18 (d, 2H, J = 6.1 Hz), 3.56 (q, 2H, J = 6.2 Hz), 4.10 (d, 2H, J =
5.2 Hz), 5.77 (dt, 1H, J = 15.5, 6.1 Hz), 5.86 (dt, 1H, J = 15.5, 5.2 Hz), 7.81 (d, 1H, J = 8.8 Hz), 8.04
(dd, 1H, J = 1.6, 8.8 Hz), 8.33 (m, 1H), 8.57 (d, 1H, J = 1.6 Hz), 9.60 (s, 1H). 13C NMR (CDCl3) δ 12.1,
37.5, 48.1, 51.6, 55.6, 63.2, 98.1, 128.8, 130.8, 132.7, 138.6, 139.6, 139.8, 144.0, 144.4, 144.6, 163.1.
ESIꢀMS m/z [M + H]+ 441.13.
General Procedure: Syntheses of mesylates 22, 25, 28, 29 and 31. To a solution of the corresponding
alcohol, distilled triethylamine (1.2 equiv) and DMAP (0.1 equiv) in anhydrous DCM (1ꢀ2 mL) was
added MsCl (1.2 equiv). The mixture was stirred at rt for 30ꢀ120 min and then poured into a saturated
aqueous sodium hydrogencarbonate solution (10ꢀ30 mL). After decanting, the aqueous layer was
extracted with ethyl acetate (3 x 15 mL). The organic layers were combined, washed with brine
(15 mL), dried on MgSO4, filtered and evaporated under reduced pressure to afford the crude product.
N-[2-[[N-Ethyl-N-(4-methanesulfonyloxybut-2-ynyl)]amino]ethyl]-6-iodoquinoxaline-2-carboxamide
(22). The alcohol 21 (25 mg, 57 ꢁmol) was treated according to the general method described above
(reaction time: 60 min) and purified by chromatography (SiO2, ethyl acetate) to give compound 22
(23 mg, 45 ꢁmol) as a yellow gum. Yield 78%; Rf (SiO2, ethyl acetate) 0.31; IR (NaCl) ν 732, 907,
1025, 1177, 1370, 1474, 1523, 1674, 2254, 2869, 2928, 3200ꢀ3600. 1H NMR (CDCl3) δ 1.12 (t, 3H, J =
7.1 Hz), 2.64 (q, 2H, J = 7.1 Hz), 2.81 (t, 2H, J = 6.1 Hz), 3.10 (s, 3H), 3.59 (s, 2H), 3.62 (q, 2H, J =
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Journal of Medicinal Chemistry
6.1 Hz), 4.88 (t, 2H, J = 1.8 Hz), 7.81 (d, 1H, J = 8.9 Hz), 8.07 (dd, 1H, J = 8.9, 1.9 Hz), 8.25 (m, 1H),
8.59 (d, 1H, J = 1.9 Hz), 9.62 (s, 1H). 13C NMR (CDCl3) δ 12.9, 37.0, 38.9, 41.8, 47.9, 52.1, 57.6, 80.2,
83.9, 98.2, 130.8, 138.7, 139.5 (2C), 139.8, 143.9 (2C), 144.4 (2C), 144.7, 163.1. ESIꢀMS m/z [M + H]+
517.04.
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2
3
4
5
6
7
8
9
N-[2-[N-Ethyl-N-[(E)-4-methanesulfonyloxybut-2-enyl]amino]ethyl]-6-iodoquinoxaline-2-
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carboxamide (25). The alcohol 24 (25.5 mg, 58 ꢁmol) was treated according to the general method
described above (reaction time: 30 min) and purified by chromatography (SiO2, ethyl acetate) to give
compound 25 (21 mg, 41 ꢁmol) as a yellow gum. Yield 70%; Rf (SiO2, ethyl acetate) 0.35; IR (NaCl) ν
1
409, 828, 927, 1172, 1352, 1474, 1527, 1592, 1670, 2858, 2929, 3300–3400 cmꢀ1. H NMR (CDCl3) δ
1.09 (t, 3H, J = 7.1 Hz), 2.63 (q, 2H, J = 7.1 Hz), 2.73 (t, 2H, J = 6.1 Hz), 2.99 (s, 3H), 3.21 (m, 2H),
3.58 (q, 2H, J = 6.1 Hz), 4.69 (m, 2H), 5.88 (m, 2H), 7.84 (d, 1H, J = 8.9 Hz), 8.09 (dd, 1H, J = 8.9,
1.7 Hz), 8.36 (m, 1H), 8.61 (d, 1H, J = 1.7 Hz), 9.64 (s, 1H). 13C NMR (CDCl3) δ 12.1, 37.3, 38.1, 47.8,
51.8, 54.9, 69.8, 98.0, 127.3, 130.9, 132.6, 138.6, 139.8, 144.7, 162.9. ESIꢀMS m/z [M + H]+ 519.07
N-(12-ethyl-1-methanesulfonyloxy-3,6,9-trioxa-12-azatetradecan-14-yl)-6-iodoquinoxaline-2-
carboxamide (28). The alcohol 26 (80 mg, 146 ꢁmol, prepared as described by Maisonial, Billaud et
al.38) was treated according to the general method described above (reaction time: 60 min) and purified
by chromatography (SiO2, DCM → DCM/EtOH, 9:1, v/v) to give compound 28 (85 mg, 136 ꢁmol) as a
yellow gum. Yield 93%; Rf (SiO2, DCM/EtOH, 9:1, v/v) 0.37; IR (ATR diamond accessory) ν 751, 917,
1
1106, 1171, 1349, 1473, 1592, 1525, 1668, 2849, 2916, 3300ꢀ3400 cmꢀ1. H NMR (CDCl3) δ 1.12 (t,
3H, J = 7.1 Hz), 2.85 (m, 6H), 3.06 (s, 3H), 3.60 (m, 12H), 3.73 (m, 2H), 4.36 (m, 2H), 7.83 (d, 1H, J =
13
8.8 Hz), 8.06 (dd, 1H, J = 8.8, 1.8 Hz), 8.53 (m, 1H), 8.60 (d, 1H, J = 1.8 Hz), 9.62 (s, 1H). C NMR
(CDCl3) δ 11.7, 37.2, 37.8, 48.9, 52.9, 53.0, 69.1, 69.3, 70.5 (5C), 98.1, 130.9, 138.6, 139.6, 139.8,
144.1, 144.4, 144.7, 163.2. ESIꢀMS m/z [M + H]+ 625.25.
N-(24-ethyl-1-methanesulfonyloxy-3,6,9,12,15,18,21-heptaoxa-24-azahexacosan-26-yl)-6-
iodoquinoxaline-2-carboxamide (29). The alcohol 27 (91 mg, 126 ꢁmol, prepared as described by
Maisonial, Billaud et al.38) was treated according to the general method described above (reaction time:
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120 min) and purified by chromatography (SiO2, DCM → DCM/EtOH, 9:1, v/v) to give compound 29
(95 mg, 119 ꢁmol) as a yellow gum. Yield 95%; Rf (SiO2, DCM/EtOH, 8:2, v/v) 0.50; IR (ATR
diamond accessory) ν 733, 923, 1128, 1176, 1354, 1474, 1593, 1527, 1675, 2800ꢀ3000, 3300ꢀ3400 cmꢀ
1. 1H NMR (CDCl3) δ 1.04 (t, 3H, J = 7.1 Hz), 2.64 (q, 2H, J = 7.1 Hz), 2.73 (m, 4H), 3.06 (s, 3H), 3.61
(m, 28H), 3.73 (t, 2H, J = 4.5 Hz), 4.35 (t, 2H, J = 4.5 Hz), 7.80 (d, 1H, J = 8.8 Hz), 8.05 (dd, 1H, J =
8.8, 1.3 Hz), 8.41 (m, 1H), 8.57 (d, 1H, J = 1.3 Hz), 9.61 (s, 1H). 13C NMR (CDCl3) δ 12.2, 37.6, 37.8,
48.7, 52.9, 53.0, 69.1, 69.4, 70.2ꢀ70.6 (13C), 98.0, 130.9, 138.6, 139.6, 139.7, 144.2, 144.4, 144.7,
163.0. ESIꢀMS m/z [M + H]+ 801.33.
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N-[2-[N-Ethyl-N-(3-methanesulfonyloxypropyl)amino]ethyl]-6-iodoquinoxaline-2-carboxamide (31)
.
The alcohol 30 (30 mg, 70 ꢁmol, prepared as described by Maisonial, Billaud et al.38) was treated
according to the general method described above (reaction time: 60 min) and purified by
chromatography (SiO2, DCM → DCM/EtOH, 9:1, v/v) to give compound 31 (32.5 mg, 64 ꢁmol) as a
1
yellow gum. Yield 92%; Rf (SiO2, DCM/EtOH, 9/1, v/v) 0.43; H NMR (CDCl3) δ 1.07 (t, 3H, J =
7.1 Hz), 1.91 (quint., 2H, J = 6.4 Hz), 2.65 (m, 6H), 2.97 (s, 3H), 3.58 (q, 2H, J = 5.9 Hz), 4.37 (t, 2H, J
= 6.2 Hz), 7.86 (d, 1H, J = 8.9 Hz), 8.08 (dd, 1H, J = 8.9, 1.9 Hz), 8.32 (m, 1H), 8.60 (d, 1H, J =
1.9 Hz), 9.62 (s, 1H). 13C NMR (CDCl3) δ 11.8, 27.3, 37.3, 37.5, 47.4, 49.2, 52.3, 67.9, 98.2, 131.0,
138.4, 139.6, 139.8, 144.0, 144.5, 144.6, 163.0. ESIꢀMS m/z [M + H]+ 507.08.
Radiochemistry
[18F]KF,K222 complex. The activated [18F]KF,K222 complex was obtained by usual method in 19 min.
Briefly, the aqueous [18F]Fꢀ solution from target was flushed through an anion exchange resin (SepꢀPak
Light Accell Plus QMA cartridge, Waters). The radioactivity was eluted to the reactor with a K2CO3
(3.0 mg) and Kryptofix (K222, 15.0 mg) solution (1 mL, MeCN/H2O, 70:30, v/v). Solvents were
evaporated under reduced pressure and under a gentle stream of helium at 110 °C for 10 min. A second
azeotropic drying was repeated by addition of anhydrous MeCN (1.0 mL).
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Journal of Medicinal Chemistry
[18F]N-[2-[N-Ethyl-N-(2-fluoroethyl)amino]ethyl]-6-iodoquinoxaline-2-carboxamide [18F]4. To the
dry [18F]KF,K222 complex were added a solution of ditosylate compound 32 in dry MeCN
(6 mg/0.5 mL) and a solution of amine 34 in dry MeCN (12 mg/1.0 mL).41 The mixture was heated at
70 °C for 10 min and then at 110 °C for 10 min. After cooling to rt, the reaction mixture was diluted
with DCM (2 mL) and directly purified by semiꢀpreparative HPLC (SemiꢀPrep SiO2 column;
DCM/EtOH 98:02 v/v; tR = 14.9 min). The solvent was evaporated under reduced pressure and the
formulation was achieved in EtOH/saline (1:9 v/v; formulation yield = 97%). [18F]4 was obtained in
91 min with 11% overall RCY (6% nonꢀdecayꢀcorrected). RCP was determined by analytical HPLC
(SiO2 column; DCM/EtOH 98:2 v/v; 1 mL.minꢀ1; tR=12.65 min) and was >99%. Radiotracer was
radiochemically stable at rt for at least 6 h after preparation.
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[18F]N-[2-[N-Ethyl-N-(3-fluoropropyl)amino]ethyl]-6-iodoquinoxaline-2-carboxamide [18F]5. To the
dry [18F]KF,K222 complex was added a solution of mesylate precursor 31 in dry MeCN (14 mg/1 mL)
and the mixture was heated at 90 °C for 10 min. After cooling to rt, the reaction mixture was diluted
with HPLC eluent (2.5 mL) and purified by semiꢀpreparative HPLC (SemiꢀPrep SiO2 column;
DCM/EtOH/NH4OH 93.8:6:0.2 v/v/v; tR = 9.4 min). The solvent was evaporated under reduced pressure
and the formulation was achieved in EtOH/saline (1:9 v/v; formulation yield = 91%). [18F]5 was
obtained in 70 min with 54% overall RCY (34% nonꢀdecayꢀcorrected). RCP was determined by
analytical HPLC (SiO2 column; DCM/EtOH/NH4OH 99.8:0:0.2 to 89.8:10:0.2 in 10 min; 1 mL.minꢀ1; tR
= 8.67 min) and was >99%. Radiotracer was radiochemically stable at rt for at least 6 h after
preparation. LogD = 1.28±0.04.
[18F]N-[2-[N-Ethyl-N-[(E)-4-fluorobut-2-enyl]amino]ethyl]-6-iodoquinoxaline-2-carboxamide
[18F]6. To the dry [18F]KF,K222 complex was added a solution of mesylate precursor 25 in dry MeCN
(12 mg/1 mL) and the mixture was heated at 90 °C for 10 min. After cooling to rt, the reaction mixture
was diluted with HPLC eluent (2.5 mL) and purified by semiꢀpreparative HPLC (SemiꢀPrep C18
column; MeOH/H2O/NH4OH 74.9:24.9:0.2 v/v/v; tR = 16.0 min). The collected solution was diluted in
saline (20 mL) then passed through a C18 cartridge (SepꢀPak Plus C18, Waters). The cartridge was then
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washed with water (5 mL) before being eluted with EtOH (1 mL) and then saline (10 mL). The solution
was finally passed through a Millipore filter (0.22 ꢁm) and collected in a sterile vial (formulation yield:
83%). [18F]6 was obtained in 62 min with 16% overall RCY (11% nonꢀdecayꢀcorrected). RCP was
determined by analytical HPLC (C18 column; MeOH/H2O/NH4OH 74.9:24.9:0.2 v/v/v; 1 mL.minꢀ1; tR
= 8.62 min) and was >99%. Radiotracer was radiochemically stable at rt for at least 6 h after
preparation. LogD = 1.64±0.02.
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[18F]N-[2-[[N-Ethyl-N-(4-fluorobut-2-ynyl)]amino]ethyl]-6-iodoquinoxaline-2-carboxamide [18F]7.
To the dry [18F]KF,K222 complex was added a solution of mesylate precursor 22 in dry MeCN
(10 mg/1 mL) and the mixture was heated at 90 °C for 10 min. After cooling to rt, the reaction mixture
was diluted with HPLC eluent (2.5 mL) and purified by semiꢀpreparative HPLC (SemiꢀPrep C18
column; MeOH/H2O/NH4OH (74.9:24.9:0.2 v/v/v; tR = 11.2 min). The collected solution was diluted
with saline (20 mL) then passed through a C18 cartridge (SepꢀPak Plus C18, Waters). The cartridge was
then washed with water (5 mL) before being eluted with EtOH (700 ꢁL) and then saline (10 mL). The
solution was finally passed through a Millipore filter (0.22 ꢁm) and collected in a sterile vial
(formulation yield: 76%). [18F]7 was obtained in 59 min with 22% overall RCY (15% nonꢀdecayꢀ
corrected). RCP was determined by analytical HPLC (C18 column; MeOH/H2O/NH4OH 74.9:24.9:0.2
v/v/v; 1 mL.minꢀ1; tR = 8.33 min) and was >99%. Radiotracer was radiochemically stable at rt for at
least 6 h after preparation. LogD = 1.90±0.02.
[18F]N-(12-ethyl-1-fluoro-3,6,9-trioxa-12-azatetradecan-14-yl)-6-iodoquinoxaline-2-carboxamide
[18F]8. To the dry [18F]KF,K222 complex was added a solution of mesylate precursor 28 in dry MeCN
(13 mg/1 mL) and the mixture was heated at 90 °C for 10 min. After cooling to rt, the reaction mixture
was diluted with HPLC eluent (2.5 mL) and purified by semiꢀpreparative HPLC (C18 column;
MeOH/H2O/NH4OH (74.7:24.9:0.4 v/v/v; tR = 12.5 min). The collected solution was diluted with saline
(20 mL) then passed through a C18 cartridge (SepꢀPak Plus C18, Waters). The cartridge was then
washed with water (5 mL) before being eluted with EtOH (900 ꢁL) and then saline (8 mL). The solution
was collected in a sterile vial (formulation yield: 90%). [18F]8 was obtained in 57 min with 21% overall
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RCY (14% nonꢀdecayꢀcorrected) and a specific activity of 45ꢀ53 GBq.ꢁmolꢀ1. RCP was determined by
analytical HPLC (C18 column; MeOH/H2O/NH4OH v/v/v; 59.9:39.9:0.2 to 89.8:10:0.2 linear gradient
in 10 min then 59.9:39.9:0.2 isocratic during 7 min; 0.9 mL.minꢀ1; tR = 12.51 min) and was >99%. Color
spot test showed no detection of K222 in the final injectable [18F]8 solution. Radiotracer was
radiochemically stable at rt for at least 22 h after preparation. LogD = 1.52±0.03.
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[18F]N-(24-ethyl-1-fluoro-3,6,9,12,15,18,21-heptaoxa-24-azahexacosan-26-yl)-6-iodoquinoxaline-2-
carboxamide [18F]9. To the dry [18F]KF,K222 complex was added a solution of mesylate precursor 29 in
dry MeCN (15 mg/1 mL) and the mixture was heated at 90 °C for 10 min. After cooling to rt, the
solution was diluted with HPLC eluent (2.5 mL) and purified by semipreparative HPLC (SiO2 column;
DCM/EtOH/NH4OH 91.8:8:0.2 v/v/v; tR = 12.1 min). The solvent was evaporated under reduced
pressure and the formulation was achieved in EtOH/saline (05:95; v/v; formulation yield: 93%). [18F]9
was obtained in 65 min with 34% overall RCY (25% nonꢀdecayꢀcorrected), and a specific activity of
81ꢀ139 GBq.ꢁmolꢀ1. RCP was determined by analytical HPLC (C18 column; MeOH/H2O/NH4OH
v/v/v; 59.9:39.9:0.2 to 89.8:10:0.2 linear gradient in 10 min then 59.9:39.9:0.2 isocratic during 7 min;
0.9 mL.minꢀ1; tR = 12.59 min) and was >99%. Color spot test showed no detection of K222 in the final
injectable [18F]9 solution. Radiotracer was radiochemically stable at rt for at least 6 h after preparation.
LogD = 0.95±0.02.
ACKNOWLEDGMENT We would like to thank the CLARA (Cancéropôle Lyon RhôneꢀAlpes
Auvergne), the Auvergne Regional Council, the Puy de Dôme General Council, Clermont Community,
the European Regional Development Fund, the European Union, The French Ligue contre le Cancer and
the Bullukian Foundation for their financial support. We also thank Martine Bayle, Laurent Audin,
Pierre Labarre (INSERM U 990/Université d’Auvergne) and Raphael Boisgard (CEA/SHFJ, Orsay,
France) for technical assistance.
ABBREVIATIONS α–MSH, αꢀmelanocyteꢀstimulating hormone; EIꢀMS, electron impact mode mass
spectra; ESIꢀMS, electrospray ionization mass spectra; FDG, fluorodeoxyglucose; MC1R, melanocortin
type 1 receptor; MsCl, methanesulfonyl chloride; p.i., post injection; RCP, radiochemical purity; RCY,
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radiochemical yield; SLNB, sentinel lymph node biopsy; TBDMSCl, tertꢀbutyldimethylsilyl chloride;
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TRT, targeted radionuclide therapy.
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Figure 1. Chemical structure of 1 (ICF01006),29 2 (ICF01012),28 and 3,37 three examples of
arylcarboxamides with high affinity for melaninꢀcontaining cells, evaluated in clinical or preclinical
studies.
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F
N
O
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O
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N
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H
H
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N
I
I
Figure 2. Chemical structures of the six iodinated and fluorinated 6ꢀiodoquinoxalineꢀ2ꢀcarboxamide
derivatives investigated.
O
N
N
N
N
H
R
I
F
4 R=
5 R=
6 R=
7 R=
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R=
9 R=
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