Synthesis, radioiodination and in vivo screening of novel potent iodinated and fluorinated radiotracers as melanoma imaging and therapeutic probes

Article abstract of DOI:10.1016/j.ejmech.2012.11.047

In order to develop new iodinated and fluorinated matched-pair radiotracers for Single-Photon Emission Computed Tomography (SPECT)/Positron Emission Tomography (PET) imaging and targeted radionuclide therapy of melanoma, we successfully synthesized and ra

Full text of DOI:10.1016/j.ejmech.2012.11.047

European Journal of Medicinal Chemistry 63 (2013) 840e853
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, radioiodination and in vivo screening of novel potent
iodinated and fluorinated radiotracers as melanoma imaging and
therapeutic probes
,
b
,
c
, ,
,
b
,
c
,
,
b
,
,
,
b
,c
Aurélie Maisonial a
1, Emilie M.F. Billaud a 1, Sophie Besse a c, Latifa Rbah-Vidal a
,
*
Janine Papon a c, Laurent Audin a c, Martine Bayle a c, Marie-Josèphe Galmier a
,
,
b,
,
b,
,
b,
,b,c
,
b
,
,
b,
,b
c
Sébastien Tarrit a c, Michèle Borel a c, Serge Askienazy d, Jean-Claude Madelmont a
,
Nicole Moins a c, Philippe Auzeloux a c, Elisabeth Miot-Noirault a
,
,
b,
,
b,
,b,c
Jean-Michel Chezal a
,b,c
a Clermont Université, Université d’Auvergne, BP 10448, F-63000 Clermont-Ferrand, France
b Inserm, UMR 990, Imagerie Moléculaire et Thérapie Vectorisée, F-63005 Clermont-Ferrand, France
c Centre Jean Perrin, F-63011 Clermont-Ferrand, France
d Laboratoires Cyclopharma, Biopôle Clermont-Limagne, Saint-Beauzire F-63360, France
a r t i c l e i n f o
a b s t r a c t
Article history:
In order to develop new iodinated and fluorinated matched-pair radiotracers for Single-Photon Emission
Computed Tomography (SPECT)/Positron Emission Tomography (PET) imaging and targeted radionuclide
therapy of melanoma, we successfully synthesized and radiolabelled with iodine-125 seven new de-
rivatives, starting from our previously described lead structure 3. The relevance of these radiotracers for
gamma scintigraphic imaging of melanoma in rodent was assessed. The tumoural radioactivity uptake
was most often high and specific even at early time points (12.1e18.3% ID/g at 3 h p.i. for [125I]39e42)
and a fast clearance from the non-target organs was observed. Also, calculated effective doses that could
be delivered to tumours when using corresponding [131I]-labelled analogues were generally higher than
100 cGy/MBq injected (98.9e150.5 cGy/MBq for [131I]39e42). These results make compounds 39e42
suitable candidates for (i) PET imaging of melanoma after labelling with fluorine-18 and (ii) targeted
radionuclide therapy of disseminated melanoma after labelling with iodine-131.
Received 14 June 2012
Received in revised form
14 November 2012
Accepted 17 November 2012
Available online 24 March 2013
Keywords:
Melanoma
SPECT imaging
Iodinated and fluorinated radiotracers
Iodine-125
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
worldwide [1e4]. In the United States the estimated lifetime risk of
developing cutaneous melanoma is currently 1 in 58 overall against
In contrast to many other malignancies, the incidence of mela-
noma, the most lethal form of skin cancer, is steadily increasing
1 in 250 in 1980 [5,6]. Survival largely depends on the stage of
detection [7,8]. At the earliest invasive stages, melanoma is usually
cured by surgical excision and the estimated 5-year overall survival
rate is 97% [6]. Unfortunately, this cancer can rapidly spread to
almost all the organs, and for patients with advanced-stage disease,
the survival rate decreases dramatically (12% at 5 years) [9]. This
poor prognosis is due to the ineffectiveness of existing chemo- or
immunotherapies [6,9,10]. In addition, although promising results
in terms of responses to treatment are obtained with new targeted
therapy approaches, median progression-free survival is still
disappointingly low [11e13]. Powerful tools for both early detection
and new efficient treatment strategies of melanoma are therefore
urgently needed.
Abbreviations: DAFBA, N-(2-diethylaminoethyl)-4-fluorobenzamide; DAST,
(diethylamino)sulphur trifluoride; DME, dimethoxyethane; ESI-MS, electrospray
ionization mass spectra; ID, injected dose; i.v., intravenous injection; PET, positron
emission tomography; p.i., post injection; RCP, radiochemical purity; RCY, radio-
chemical yield; ROI, region of interest; RP-HPLC, reverse-phase high performance
liquid chromatography; SA, specific activity; SPECT, single-photon emission
computed tomography; TBDMSCl, tert-butyldimethylsilyl chloride; TBAF, tetrabu-
tylammonium fluoride.
*Corresponding authors. UMR 990 Inserm/UdA, IMTV, 58, rue Montalembert,
BP 184, F-63005 Clermont-Ferrand, France. Tel.: þ33 4 73 15 08 00; fax: þ33
4 73 15 08 01.
An interesting area of melanoma research is the development of
radiopharmaceuticals specific to this pathology. Among all the
E-mail address: aurelie.maisonial@inserm.fr (A. Maisonial).
Both authors contributed equally.
1
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.11.047

A. Maisonial et al. / European Journal of Medicinal Chemistry 63 (2013) 840e853
841
approaches investigated, melanins are relevant molecular targets
duetothecapacityof these amorphous, irregular natural polymersto
bind with many drugs, and especially those with coplanar fused ar-
omatic rings [14]. Based on the work done on the benzamide family
[15e20], recent studies using radiohalogenated heteroaromatic an-
alogues have revealed that this class of compounds is suitable for
positron emission tomography (PET) imaging (e.g. [18F]1, [18F]DAFBA
[21e26]) or targeted radionuclide therapy (e.g. [131I]2, [131I]MIP-
1145) of melanoma (Fig. 1) [27e30]. These findings prompted us to
investigate a new approach consisting in using iodinated and fluo-
rinated matched-pair radiotracers targeting melanins and offering
potential for both diagnosis via single-photon emission computed
tomography (SPECT, iodine-123) or PET imaging (iodine-124 or
fluorine-18), and therapy (iodine-131), depending on the type of
radionuclide introduced on the same chemical scaffold [31].
Fig. 2. Pharmacomodulation of the fluoro side chain of 3.
Fourteen iodinated compounds bearing a 2- or 6-fluoropyridine
moiety on the N,N-diethylethylenediamine side chain were
recently synthesized, radiolabelled with iodine-125 and screened
by gamma scintigraphic imaging to evaluate their in vivo bio-
distribution profiles in B16F0 primary melanoma-bearing mice
[31,32]. This screening allowed the selection of the tracer 3 (Fig. 1)
with high, specific and long-lasting tumoural uptake. This com-
pound was then radiolabelled with fluorine-18 in a three-step
procedure. A first PET imaging experiment using the same mu-
rine model confirmed the promising results obtained by gamma
scintigraphic imaging, and the utility of combining such tracer
specificity with the performance of PET technology. Compound 3
was finally radiolabelled with iodine-131 and evaluated in B16F0
primary melanoma-bearing mice for a first targeted radionuclide
therapy assay. This treatment induced a significant tumoural
growth inhibition. Promising properties of 3 radiolabelled with
either fluorine-18 or iodine-131 gave a first validation of our
concept and underscored the potential of this class of iodinated and
fluorinated matched-pair radiotracers for both diagnosis and tar-
geted radionuclide therapy of melanoma [32]. To further improve
pharmacokinetic profiles of the tracers, and facilitate radio-
fluorination processes, we planned to extend this strategy to fluo-
roaliphatic derivatives of the lead compound 3 as depicted in Fig. 2.
Here we describe the synthesis, and iodine-125 radiolabelling of
these new analogues and their preliminary in vivo screening in a
melanoma-bearing mice model.
mind that haloalkylamines can undergo intramolecular cyclisation
[33,34], we synthesized more constrained derivatives containing
fluoroallyl and fluoropropargyl synthons, together with pegylated
analogues enlarging the distance between the fluorine atom and
the tertiary amine. Finally, we explored the Huisgen 1,3-dipolar
cycloaddition between azides and alkynes, known as “click”
chemistry, which is recognized as a powerful synthetic methodol-
ogy allowing rapid and convenient radiofluorinations [35].
The synthesis of compounds 8 and 14, bearing fluoroethyl or
fluoropropyl side chains respectively, is illustrated in Scheme 1. First,
alcohol 4 [32] was converted into the fluorinated compound 5 using
(diethylamino)sulphur trifluoride (DAST) at ꢀ50 ꢁC. Deprotection of
the phthalimide 5 was achieved by the action of hydrazine mono-
hydrate to give unstable primary amine 6, which was rapidly
condensed with p-nitrophenyl 6-iodoquinoxaline-2-carboxylate (7)
[36] to provide the desired amide 8. A modified synthetic pathway
was designed to produce the fluoropropyl derivative 14 in order to
introduce the fluorine atom as late as possible in the synthesis.
Phthalimide 9 [37] was alkylated with commercially available (3-
bromopropoxy)-tert-butyldimethylsilane to afford compound 10. In
a similar manner as for 6, phthalimide 10 was then deprotected to
give primary amine 11 in quantitative yield and coupled to the acti-
vated ester 7 to provide amide 12. Desilylation of 12 using tetrabu-
tylammonium fluoride (TBAF) afforded alcohol 13, which was finally
converted into the desired fluorinated compound 14 following the
protocol optimized for 5. We note that due to its instability, tracer 14
has to be synthesized and purified just before use.
To synthesize amides 17 and 20 (Scheme 2) we investigated a
direct alkylation of N-[2-(ethylamino)ethyl]-6-iodoquinoxaline (15)
[36] using either (E)-4-fluoro-but-2-enyl toluene-4-sulfonate (16)
[38] or 4-fluoro-but-2-ynyl toluene-4-sulfonate (19). The latter was
produced via atwo-step reactionsequenceaccording tothe procedure
developed for compound 16 involving ditosylation [39] of commer-
cially available 1,4-butynediol, and subsequent monofluorination [38]
of 18 with TBAF in refluxing tetrahydrofuran. This strategy afforded
amides 17 and 20 with moderate yields of 47% and 38% respectively.
The (E)-configuration of final alkene 17 was extrapolated from the
vicinal spinespin coupling constant of 15.8 Hz between the two
olefinic protons of the corresponding dihydrochloride salt 39.
2. Results and discussion
2.1. Chemistry
The synthesis of seven new derivatives showing structural
similarity to 3 was undertaken, based on the replacement of the (2-
fluoropyridin-3-yloxy)ethyl moiety by various fluoroaliphatic
groups (Fig. 2). As a first approach, we designed compounds
bearing saturated fluoroalkyl chains. In parallel, and keeping in
Pegylated derivatives 33 and 34 were synthesized by a common
strategy outlined in Scheme 3. Commercially available tetra- and
octaethylene glycols were first monoprotected using tert-butyldi-
methylsilyl chloride (TBDMSCl) according to a slightly modified
protocol developed by Kan and co-workers [40]. Iodination of the
remaining free alcohol function of 21 and 22, in the presence of
triphenylphosphine and imidazole, afforded derivatives 23 and 24.
Subsequent nucleophilic substitution using phthalimide 9 [37]
provided intermediates 25 and 26. Under similar reaction condi-
tions (temperature and reaction time), we observed that two
equivalents of potassium carbonate were required to obtain 26 in
Fig. 1. Several benzamide derivatives developed for imaging and/or targeted radio-
nuclide therapy of melanoma.

842
A. Maisonial et al. / European Journal of Medicinal Chemistry 63 (2013) 840e853
Scheme 1. Syntheses of compounds 8 and 14. Reagents and conditions: (i) DAST, DME, ꢀ50 ꢁC, 15 min, then rt, 1.5 h; (ii) NH2NH2$H2O, EtOH, reflux, 1 h; (iii) THF, rt, overnight; (iv)
K2CO3, CH3CN, 50 ꢁC, 36 h; (v) NH2NH2$H2O, EtOH, rt, 18 h; (vi) TBAF, THF, rt, 1.5 h.
65% yield, whereas only one equivalent was needed to produce 25
(72%). Deprotection of phthalimides 25 and 26 using hydrazine
monohydrate in ethanol immediately followed by peptidic
coupling with activated ester 7 [36] provided silylated derivatives
29 and 30, respectively. After desilylation with TBAF, the resulting
alcohols 31 and 32 were converted into the corresponding alkyl
fluorides 33 and 34 using DAST as fluorinating reagent.
The sequence depicted in Scheme 4 illustrates the synthesis of
amide 37, obtained by coupling alkyne 35 and azide 36 using cop-
per(I) generated in situ as catalyst. Alkyne 35 was synthesized
through alkylation of compound 15 with propargyl bromide. Un-
stable 1-azido-2-fluoroethane (36) wasproduced in situ by the action
of freshly dried cesium fluoride on 2-azidoethyl p-toluenesulfonate
[41] according to the procedure described by Bejot et al. [42].
To improve the solubility of final compounds in water, hydro-
chloride salts 38e42 were prepared in 78e89% yields by treatment
of corresponding amides 8, 17, 20, 33 and 37 respectively, with an
anhydrous 2 N HCl/ether solution (Table 1). However, in the case of
derivatives 14 and 34, the conversion into hydrochloride salts failed
due to unexpected chlorine-for-fluorine nucleophilic substitution.
2.2. Radiochemistry
Derivatives 14, 34, 38e42 were radioiodinated at low specific
activity using a nucleophilic aromatic isotopic exchange reaction
with no-carrier-added [125I]NaI in the presence of copper sulphate
and under acidic conditions (Table 2). The radiolabelling process for
each radiotracer was optimized with respect to the following re-
action parameters: temperature, solvent and reaction time. The
highest radiolabelling efficiencies were usually achieved after 25e
45 min heating at 130 ꢁC in either acetic acid solution (method A) or
citrate buffer (pH ¼ 4, method B). According to radio-thin layer
chromatography (radio-TLC) and radio-reversed phase-high pres-
sure liquid chromatography (radio-RP-HPLC) analyses of the reac-
tion mixtures, only a limited number of by-products were produced
when using these optimized reaction parameters. After purification
on an ExtrelutÒ column and then semi-preparative RP-HPLC, ex-
pected radiotracers [125I]14, [125I]34 and [125I]38e42 were pro-
duced with moderate radiochemical yields (RCY, 8e50%), high
radiochemical purities (RCP, >96%) and with a specific activity (SA)
range of 4.0e39.6 MBq/mmol (Table 2).
Scheme 2. Syntheses of compounds 17 and 20. Reagents and conditions: (i) CH3CN, rt, 60 h; (ii) p-toluenesulfonyl chloride, KOH, Et2O, e15 ꢁC then 0 ꢁC, 2 h; (iii) TBAF, THF, reflux,
2 h; (iv) CH3CN, rt, 72 h.

A. Maisonial et al. / European Journal of Medicinal Chemistry 63 (2013) 840e853
843
Scheme 3. Syntheses of pegylated derivatives 33 and 34. Reagents and conditions: (i) (a) NaH, THF, 0 ꢁC, 5 min; (b) TBDMSCl, rt, 1 or 4 h; (ii) imidazole, Ph3P, I2, CH2Cl2, 0 ꢁC, 30 min
and then rt, 1 h or 48 h; (iii) K2CO3, CH3CN, 50 ꢁC, 6 or 7 d; (iv) NH2NH2$H2O, EtOH, rt, 20 h; (v) THF, rt, 21 h; (vi) TBAF, THF, rt, 1.5 h or 2 h; (vii) DAST, DME, e50 ꢁC, 15 min then rt,
1.5 h.
2.3. In vivo screening in melanoma-bearing mice
values were observed at early time points after injection of [125I]40
or [125I]3, but from 24 h p.i. a significantly higher retention of
radioactivity in the tumour was noted for [125I]40 (11.6 ꢂ 1.7% ID/g
for [125I]40 vs. 5.2 ꢂ 1.2% ID/g for [125I]3 at 5 days p.i.). Bio-
distribution profiles of [125I]14 and [125I]39e42 are promising and
consistent for PET imaging purposes. However, due to its chemical
instability, derivative 14 bearing a 3-fluoropropyl side chain
appeared less attractive than compounds 39e42. According to us,
and in regard to melanoma uptake, [125I]39e42 were the most
promising and non toxic radiotracers. This was confirmed by the
high tumour to muscle ratios determined for these compounds as
early as 1 h p.i. (in the range of 5.76 ꢂ 1.22e7.84 ꢂ 2.83).
The pharmacological evaluation of all final radiotracers was
performed according to the standard protocol established by Miot-
Noirault et al. [43]. The in vivo gamma imaging screening of the
[
125I]-labelled radiotracers was carried out in a B16F0-bearing
mouse model and high tumoural uptake was globally observed
(Table 3). For all the radiotracers tested, tumour accumulation was
clearly evidenced from the first gamma scintigrams obtained at 1 h
post injection (p.i.) (Fig. 3). Also, the tumours remained visible for
at least 10 days after injection of the radioactive compounds. At
early time points, radioactivity was mainly localized in the
abdominal region, consistent with the previously observed elimi-
nation of this class of compounds via the urinary and hepatobiliary
systems [28]. Tumours were visualized with a high contrast due to a
rapid clearance of the radioactivity from the non-target organs (e.g.
Fig. 3, 24 h p.i. scintigram).
To provide further data on the potential of this new class of
compounds for targeted radionuclide therapy of melanoma, we
also estimated the dosimetry parameters of the corresponding
[
131I]-labelled analogues. Based on the radioactive concentration
values calculated from planar scintigraphic images, effective
radiation doses that could be delivered to melanoma tumours
were calculated using an adaptation of the MIRD software to the
mouse and expressed as cGy/injected MBq (Table 4). Compared
with [131I]3 (106.4 cGy/injected MBq), the estimated effective
doses were slightly lower for compounds [131I]14, [131I]34 and
Compared with our reference radiotracer [125I]3, a significantly
higher uptake of radioactivity in the melanoma tumour was
observed with [125I]38 (Table 3, entry 4: 21.4 ꢂ 4.9% injected dose/
gram (ID/g) vs. entry 1: 12.7 ꢂ 2.3% ID/g for [125I]3 at 1 h p.i.).
Unfortunately, [125I]38 administered by the intravenous (i.v.) route
proved highly toxic, with only one mouse out of six surviving 2
days after injection. By contrast, compound [125I]34 exhibited a
lower tumoural uptake than [125I]3 (Table 3, entry 3). With regard
to radiotracers [125I]14 and [125I]39e42, specific accumulation of
the radioactivity in B16F0 melanoma tumours, at least comparable
or higher than [125I]3 was evidenced. Interestingly, similar uptake
[
131I]39 (93.0, 58.9 and 98.9 cGy/injected MBq respectively).
However, promising results were obtained with tracers [131I]38,
131I]40 and [131I]42 (164.8, 140.1 and 150.5 cGy/injected MBq
[
respectively). The absorbed doses would be respectively 1.55,
1.32 and 1.41 times higher than when using [131I]3, our previous
lead tracer.
Scheme 4. Synthesis of compound 37. Reagents and conditions: (i) propargyl bromide, NEt3, EtOH, 72 h, rt. (ii) (a) CsF, 2-azidoethyl p-toluenesulfonate [41], tert-amyl alcohol,
CH3CN, 100 ꢁC, 2 h; (b) 1.0 M aq. CuSO4. 5H2O, 1.0 M aq. ascorbic acid, rt, 12 h.

844
A. Maisonial et al. / European Journal of Medicinal Chemistry 63 (2013) 840e853
Table 1
Syntheses of hydrochloride salts 38e42.
Entry
Starting matérial
R
Reaction time at rt
Product
x
Yield (%)
1
2
3
8
14 h
12 h
12 h
38
39
40
2
2
2
78
89
81
17
20
4
5
33
37
5 min
12 h
41
42
2
3
83
86
3. Conclusion
the affinity of this class of compounds for melanoma in vivo and
even enhanced the uptake of radioactivity in the tumour in some
cases. We consider that tracers 39e42 hold great potential for PET
imaging of melanoma due to their high and specific accumulation
in the murine B16F0 tumours even at early time points after in-
jection (12.1e18.3% ID/g at 3 h p.i. and 12.1e23.3% ID/g at 6 h p.i. for
In the present manuscript, we report on the synthesis, radio-
labelling with iodine-125 and in vivo screening using gamma
scintigraphic imaging of seven new iodinated and fluorinated
matched-pair radiotracers for SPECT/PET imaging and targeted
radionuclide therapy of melanoma. This investigation revealed that
the replacement of the (2-fluoropyridin-3-yloxy)ethyl side chain of
our lead structure 3 by different fluoroaliphatic moieties preserved
[
125I]39e42). With this end in view, radiolabelling of these com-
pounds with fluorine-18 and their preclinical evaluation as PET
imaging probes are currently under way in our laboratory. In
Table 2
Labelling conditions and radiochemical data for [125I]14, [125I]34, and [125I]38e42.
Entry
Starting matérial
R
Product
Method
Reaction time (min)
HPLC Rt (min)a
RCY (%)b
RCP (%)c
SA (MBq/m
mol)d
1
2
14
34
[
125I]14
125I]34
Ae
Be
30
25
17.4
13.5
16
22
>99.0
>99.0
4.0
[
15.6
3
4
5
38
39
40
[
[
[
125I]38
125I]39
125I]40
A
B
B
30
30
40
10.9
14.2
15.5
33
34
8
97.2
98.3
96.3
39.6
22.9
5.2
6
41
42
[
125I]41
125I]42
B
B
25
45
15.6
11.6
50
35
>99.0
19.3
34.6
7
[
98.8
a
HPLC Rt: HPLC retention time.
b
c
Radiochemical yields (RCY) were calculated by dividing the radioactivity of the final product by the initial amount of radioactive sodium iodide introduced.
Radiochemical purities (RCP) were assessed by RP-HPLC analyses.
SA: specific activity.
d
e
The final conversion step for the synthesis of corresponding hydrochloride salt has not been performed.

A. Maisonial et al. / European Journal of Medicinal Chemistry 63 (2013) 840e853
845
Table 3
Uptake of radioactivity in melanoma B16F0 tumours at different time points after i.v. injection of [125I]14, [125I]34, and [125I]38e42 in C57BL/6J mice. Data for [125I]3 were
already published [32] and included for comparison. Radioactive concentration values were calculated from planar scintigraphic images [43].
% ID/g ꢂ SD in B16F0 tumour
Entry
Cpd
1 h
3 h
6 h
24 h
72 h
5 d
7 d
10 d
14 d
1
2
3
4
5
6
7
8
[
[
[
[
[
[
[
[
125I]3
12.7 ꢂ 2.3b
10.0 ꢂ 2.8c
6.8 ꢂ 1.8c
20.2 ꢂ 4.2b
13.5 ꢂ 0.4c
7.8 ꢂ 1.5c
18.8 ꢂ 1.6b
15.3 ꢂ 0.7c
7.2 ꢂ 1.1c
18.2 ꢂ 0.6b
11.4 ꢂ 3.0b
6.8 ꢂ 1.7c
10.3 ꢂ 1.7b
8.5 ꢂ 1.6a
3.8 ꢂ 0.7c
20.0
5.2 ꢂ 1.2b
5.4 ꢂ 0.5a
2.7 ꢂ 0.3c
8.4
3.2 ꢂ 0.4b
3.0 ꢂ 0.1a
1.5 ꢂ 0.2b
n.d.
1.7 ꢂ 0.4b
1.4 ꢂ 0.2a
0.8 ꢂ 0.3b
1.6
0.6 ꢂ 0.3b
0.8 ꢂ 0.1a
0.3 ꢂ 0.01a
0.6
125I]14
125I]34
125I]38
125I]39
125I]40
125I]41
125I]42
21.4 ꢂ 4.9d
10.8 ꢂ 2.3b
12.3 ꢂ 4.9b
11.7 ꢂ 2.2f
13.0 ꢂ 1.5b
22.5 ꢂ 3.6d
12.3 ꢂ 2.8b
18.3 ꢂ 1.5b
12.1 ꢂ 1.7e
14.5 ꢂ 0.1b
25.0 ꢂ 8.0d
13.9 ꢂ 1.3b
23.3 ꢂ 3.5b
12.1 ꢂ 1.9e
15.9 ꢂ 1.7b
24.8 ꢂ 6.5d
15.0 ꢂ 0.5b
23.5 ꢂ 1.4b
12.4 ꢂ 2.3e
15.6 ꢂ 3.4b
10.0 ꢂ 0.9b
14.9 ꢂ 1.8b
8.6 ꢂ 3.6e
11.0 ꢂ 0.7b
5.6ꢂ1b
3.3 ꢂ 0.6b
8.0 ꢂ 0.2b
2.7 ꢂ 0.8e
5.3 ꢂ 0.8b
1.9 ꢂ 0.3b
4.2 ꢂ 0.2b
1.3 ꢂ 0.4e
3.7 ꢂ 0.3b
1.3 ꢂ 0.6b
2.2 ꢂ 0.2b
0.6 ꢂ 0.3e
1.8 ꢂ 0.4b
11.6 ꢂ 1.7b
5.8 ꢂ 2.4e
8.6 ꢂ 0.4b
n.d. non determined.
a
n ¼ 2.
b
n ¼ 3.
c
n ¼ 4.
d
n ¼ 6.
e
n ¼ 7.
f
n ¼ 8.
addition, with a fast clearance from non-target organs and very
favourable dosimetry parameters, they are also new attractive
candidates for efficient targeted radionuclide therapy of the
disseminated disease and will be evaluated for this application.
conducted under dry argon. 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, nynhydrin or potassium permanganate.
Column chromatography was performed on silica gel 60A normal
phase, 35e70
mm (Merck or SDS) or neutral aluminium oxide 90
4. Experimental section
standardized, 63e200
mm
(Merck, column chromatographic
adsorption analysis acc. to Brockmann). Uncorrected melting points
(mp) were recorded on an electrothermal IA9300 (capillary) or a
Reichert-Jung-Koffler apparatus. NMR spectra (400 or 200 MHz for
1H and 100 or 50 MHz for 13C) were recorded on a Bruker Avance
400 or 200 instrument; 19F NMR spectra (470 MHz) were recorded
on a Bruker DRX 500 apparatus using tetrafluorotoluene as internal
4.1. Materials and methods
4.1.1. Chemistry
All reagents and solvents were purchased in the following
commercial suppliers: Sigma Aldrich, Acros Organics, Carlo Erba
and SDS. All solvents were dried using common techniques [44].
Unless otherwise noted, moisture sensitive reactions were
reference (ꢀ63 ppm).
d were expressed in ppm. Infrared spectra
Fig. 3. Representative planar gamma scintigraphic images of a C57BL/6J mouse bearing a B16F0 melanoma tumour after i.v. injection of [125I]40 (3.7 MBq); T: tumour; H: head.

846
A. Maisonial et al. / European Journal of Medicinal Chemistry 63 (2013) 840e853
¼ 254 nm for [125I]41 or flow
Table 4
H2O/MeOH (30:70) (NH4OH 0.2%),
l
Tumoural dosimetry parameters calculated from the MIRD software for corre-
sponding radiotracers labelled with iodine-131.
rate ¼ 2.5 mL/min, H2O/MeOH (25:75) (NH4OH 0.2%),
l
¼ 254 nm
for [125I]34. All radiotracers were shown by radio-TLC and radio-RP-
HPLC to be identical to the authentic non-radioactive material and
to be free of significant chemical and radiochemical impurities.
Delivered dose
Entry
Cpd
cGy/injected MBq
1
2
3
4
5
6
7
8
[
[
[
[
[
[
[
[
131I]3
106.4
93.0
58.9
164.8
98.9
140.1
103.5
150.5
131I]14
131I]34
131I]38
131I]39
131I]40
131I]41
131I]42
4.1.3. Primary murine B16F0 melanoma model
Protocols were performed under the authorization of the French
“Direction des Services Vétérinaires” (authorization no. C63-113-
10) and conducted under the supervision of authorized in-
vestigators in conformity with the institution’s recommendations
for the use of laboratory animals. Animals were handled and cared
in accordance with the guidelines for the Care and Use of Labora-
tory Animals (National Research Council, 1996) and European
Directive 86/609/EEC.
C57BL/6J male mice (6e8 weeks old) were obtained from
Charles River (l’Arbresle, France) and the B16F0 syngenic mela-
noma from ATCC (no. CRL-6322). Primary melanoma model was
induced by subcutaneous injection of B16F0 murine melanoma
cells in C57BL/6J mice. B16F0 melanoma cells were cultured as
described previously [32]. Briefly, B16F0 melanoma cell cultures
were maintained as monolayers in Dulbecco’s Modified Eagle’s
Medium (DMEM)/Glutamax (Invitrogen, Cergy Pontoise, France)
supplemented with 10% foetal calf serum (Sigma, Saint Quentin
Fallavier, France), 1% vitamins (Invitrogen, Cergy-Pontoise, France),
1 mM sodium pyruvate (Invitrogen), 1% non essential amino acids
(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 (MS) were obtained on a 5989A
instrument (Agilent Technologies) or HP5890 series II chromato-
graph coupled to HP5985B mass spectrometer. The analysis of
samples was performed in CH3CN at a final concentration of
1 pmol/mL. Electrospray ionization mass spectra (ESI-MS) were
recorded on TSQ 7000 ThermoQuest Finnigam (Les Ulis, France) or
Esquire-LC (Bruker Daltonics, Wissenbourg, France) spectrometers.
The analysis of samples was performed in CH3CN at a final con-
centration of 1 pmol/mL, CH3OH/H2O (1/1, v/v, containing 1%
HCOOH) or CH3CN/H2O (1/1, v/v, containing 1% HCOOH) in positive
mode and CH3OH/H2O (1/1, v/v, containing 1% NH4OH) in negative
(Invitrogen), and 4
mg/mL of gentamycin base (Invitrogen). Cells
mode, at a final concentration of 8e12 pmol/
m
L. Each ESI-MS
were grown at 37 ꢁC in a humidified incubator containing 5% CO2.
For transplantation, cells in exponential growth phase were tryp-
sinized, washed with phosphate buffer saline (PBS), and resus-
pended in PBS. C57BL/6J mice anesthetized by isoflurane (2%)
inhalation were inoculated with 3 ꢃ 105 melanoma B16F0 cells in
0.1 mL of PBS by subcutaneous injection on the right flank.
spectrum was recorded by averaging of 10 spectra. Microanalyses
were performed by Analytical Laboratory of the CNRS (Vernaison,
France) for the elements indicated.
4.1.2. Radiochemistry
[
125I]NaI (3.7 GBq/mL, 643.8 MBq/mg) was purchased from
PerkinElmer Life and Analytical Sciences (331 Treble Cove Road,
Billerica, MA 01862, US) as a no-carrier-added solution in reductant
free 1.0 ꢃ 10ꢀ5 M aqueous sodium hydroxide solution (pH 8e11).
ExtrelutÒ and citrate buffer solution (pH ¼ 4) were purchased from
Merck (Darmstadt, Germany). The radio TLC plates (Merck neutral
aluminium oxide 60 F254 or Fluka, aluminium oxide on TLC-PET
4.2. Chemistry
4.2.1. N-[2-[N-Ethyl-N-(2-fluoroethyl)amino]ethyl]phthalimide (5)
To a solution of alcohol 4 [32] (100 mg, 0.38 mmol) in anhydrous
dimethoxyethane (DME, 7 mL) was added at ꢀ50 ꢁC, (dieth-
ylamino)sulphur trifluoride (DAST, 100 mL, 0.76 mmol) under argon.
The mixture was stirred at ꢀ50 ꢁC for 15 min and then at room
temperature for 1.5 h. Dichloromethane (5 mL) and a saturated
aqueous sodium carbonate solution (5 mL) were successively
added. The organic layer was dried on magnesium sulphate, filtered
and evaporated under vacuum. The residue was purified by chro-
matography (SiO2, ethyl acetate (EtOAc)/pentane, 1/1, v/v) to give
compound 5 (51 mg, 0.19 mmol) as a white solid. Yield 50%; Rf (SiO2,
ꢀ
foils, 60A, 0.2 mm) were developed with CH2Cl2/EtOH (98/2, v/v)
and measured on an AMBIS 400 (Scanalytics, CSPI, San Diego, CA,
USA). Analytical RP-HPLC measurements were performed on a
system consisting of a HP1100 (Hewlett Packard, Les Ulis, France)
and a Flow one A500 Radiomatic detector (Packard, Canberra,
Australia). The separation was carried out on a C18 column (Pur-
ospher RP18 e, 150 ꢃ 4.6 mm, 5
mm) using the following conditions:
gradient time ¼ 10 min, flow rate ¼ 0.5 mL/min, H2O/MeOH/
EtOAc/pentane, 1/1, v/v) 0.53; mp 57e59 ꢁC; IR (KBr)
1715, 1769, 2700e3000 cmꢀ1 1H NMR (200 MHz, CDCl3)
3H, J ¼ 7.1 Hz, CH3), 2.68 (q, 2H, J ¼ 7.1 Hz, CH2CH3), 2.86 (m, 4H,
(CO)2NCH2CH2N, NCH2CH2F), 3.78 (t, 2H, 6.6 Hz,
n
1023, 1403,
1.00 (t,
(50:50 / 0:100) (NH4OH 0.2%),
l
¼ 254 nm. For radiotracers [125I]
;
d
38, [125I]39, [125I]40 and [125I]42, semi-preparative RP-HPLC puri-
fications were performed on a system including a Shimadzu LC 6A
pump, SLC 6B controller, a CR5A integrator, a SPD 6AV UV detector
and a flow-through gamma Raytest Steffi detector. The separation
J
¼
2
(CO)2NCH2CH2N), 4.47 (td, 2H, JHeF ¼ 47.5 Hz, J ¼ 5.1 Hz, CH2F),
7.70 (m, 2H, H-5, H-6), 7.83 (m, 2H, H-4, H-7); 13C NMR (50 MHz,
was carried out on a C18 column (ZORBAX 80A, 4.6 ꢃ 150 mm) using
CDCl3)
d
11.8, 35.8, 48.5, 51.3, 53.3 (d, JCeF ¼ 21 Hz), 82.5 (d, 1JCe
2
ꢀ
the following conditions: gradient time ¼ 20 min, flow rate ¼ 1 mL/
¼ 168 Hz), 123.3 (2C), 132.2 (2C), 134.0 (2C), 168.5 (2C); 19F NMR
F
min, H2O/MeOH (50:50 / 0:100) (NH4OH 0.2%),
l
¼ 254 nm. For
(CDCl3)
d
ꢀ220.20 (tt, 3JHeF ¼ 25.0 Hz, 2JHeF ¼ 47.5 Hz); MS m/z 264
radiotracers [125I]14, [125I]34 and [125I]41 semi-preparative RP-
HPLC purification was performed on a Perkin Elmer system con-
sisting of a Flexar LC autosampler and PDA detector, a Series 200
pump, a Peltier column oven and vacuum degasser, and a GabiStar
Raytest detector. In these cases, the separation was carried out on a
(Mþ, 1), 104 (100), 76 (38), 56 (18).
4.2.2. N-(2-Aminoethyl)-N-ethyl-N-(2-fluoroethyl)amine (6)
To a stirred solution of 5 (752 mg, 2.85 mmol) in absolute
ethanol (30 mL) was added hydrazine monohydrate (324
mL,
C
18 column (SymmetryPrep C18, 7.8 ꢃ 300 mm, 7
m
¼
m, Waters) using
20 min, flow
6.68 mmol). The mixture was refluxed for 1 h. After cooling to room
temperature, the white precipitate was filtered, washed with
ethanol (10 mL) and the filtrate was evaporated under vacuum. The
residue was purified by chromatography (Al2O3, CH2Cl2/EtOH/
the following conditions: gradient time
rate ¼ 2.5 mL/min, H2O/MeOH (30:70 / 0:100) (NH4OH 0.2%),
l
¼ 254 nm for [125I]14 or isocratic elution: flow rate ¼ 3 mL/min,

A. Maisonial et al. / European Journal of Medicinal Chemistry 63 (2013) 840e853
847
NH4OH, 80/19/1, v/v/v) to give compound 6 (213 mg, 1.59 mmol) as
an orange-coloured oil. Yield 56%; Rf (Al2O3, CH2Cl2/EtOH/NH4OH,
80/19/1, v/v/) 0.41; IR (CCl4)
vacuum. The residue was suspended in dichloromethane (10 mL)
and the remaining precipitate was filtered again and washed with
dichloromethane (10 mL). The last steps were repeated until no
more white solid appeared after evaporation of the filtrate. Amine
11 (266 mg, 1.02 mmol) was obtained as a colourless oil and used
without further purification. Yield quant.; Rf (SiO2, CH2Cl2/EtOH, 9/
n ;
1034, 1453, 1684, 2700e3000 cmꢀ1
1H NMR (400 MHz, CDCl3)
d
1.01 (t, 3H, J ¼ 7.1 Hz, CH3), 2.59 (m, 4H,
3
CH2CH3, H2NCH2CH2N), 2.74 (td, 2H, JHeF ¼ 26.5 Hz, J ¼ 5.3 Hz,
CH2CH2F), 2.78 (t, 2H, J ¼ 5.3 Hz, H2NCH2CH2N), 4.30 (br s, 2H, NH2),
4.46 (td, 2H, 2JHeF ¼ 47.5 Hz, J ¼ 5.0 Hz, CH2F); 13C NMR (100 MHz,
1, v/v) 0.15; IR (NaCl)
n
776, 836, 1099, 1255, 1472, 1575, 2857, 2955,
0.04 (s, 6H,
2
CDCl3)
d
11.7, 38.8, 48.3, 53.4 (d, JCeF ¼ 20 Hz), 54.9, 82.7 (d, 1JCe
3200e3400 cmꢀ1 1H NMR (200 MHz, CDCl3)
;
d
¼ 167 Hz); 19F NMR (CDCl3)
d
ꢀ218.92; ESI-MS m/z 135.0 [M þ H]þ.
(CH3)2Si), 0.88 (s, 9H, (CH3)3C), 1.00 (t, 3H, J ¼ 7.1 Hz, CH2CH3), 1.62
F
(quint., 2H, J ¼ 6.8 Hz, NCH2CH2CH2O), 2.49 (m, 6H, CH2CH3,
4.2.3. N-[2-[[N-Ethyl-N-(2-fluoroethyl)]amino]ethyl]-6-
iodoquinoxaline-2-carboxamide (8)
NCH2CH2CH2O, (CO)2NCH2CH2N), 2.72 (t, 2H,
J
¼
6.1 Hz,
(CO)2NCH2CH2N), 3.63 (t, 2H, J ¼ 6.3 Hz, NCH2CH2CH2O); 13C NMR
To a stirred solution of amine 6 (110 mg, 0.80 mmol) in anhy-
drous tetrahydrofuran (THF, 15 mL) was added p-nitrophenyl 6-
iodoquinoxaline-2-carboxylate (7) [36] (200 mg, 0.48 mmol) un-
der argon. The mixture was stirred at room temperature overnight.
The solvent was evaporated under vacuum and the residue was
purified by chromatography (Al2O3, CH2Cl2/EtOH, 98/2, v/v) to give
compound 8 (190 mg, 0.46 mmol) as an orange-coloured oil. Yield
(50 MHz, CDCl3)
50.1, 56.8, 61.4.
d
ꢀ5.3 (2C), 11.9, 18.4, 26.0 (3C), 30.4, 39.9, 47.8,
4.2.6. N-[2-[N-Ethyl-N-[3-(tert-butyldimethylsilyloxy)propyl]
amino]ethyl]-6-iodoquinoxaline-2-carboxamide (12)
To a suspension of activated ester 7 [36] (295 mg, 0.70 mmol) in
anhydrous THF (4 mL) was added, a solution of amine 11 (283 mg,
1.09 mmol) in anhydrous THF (5 mL) under argon. The mixture was
then stirred at room temperature overnight, poured into a 1 N
aqueous sodium hydroxide solution (30 mL) and extracted with
dichloromethane (3 ꢃ 10 mL). The organic layers were combined,
dried on magnesium sulphate, filtered and evaporated under vac-
uum. The crude product was then purified by chromatography
(SiO2, CH2Cl2/EtOH, 98/2, v/v) to give compound 12 (368 mg,
0.68 mmol) as a yellow solid. Yield 97%; Rf (SiO2, CH2Cl2/EtOH, 98/2,
96%; Rf (Al2O3, CH2Cl2/EtOH, 98/2, v/v) 0.72; IR (CCl4)
n
1474, 1522,
1685, 2855, 2927 cmꢀ1 1H NMR (400 MHz, CDCl3)
;
d 1.12 (t, 3H,
J ¼ 7.1 Hz, CH3), 2.75 (q, 2H, J ¼ 7.1 Hz, CH2CH3), 2.87 (t, 1H,
3
J ¼ 6.0 Hz, (CO)NHCH2CH2N), 2.92 (td, 2H, JHeF ¼ 26.8 Hz,
J ¼ 5.0 Hz, CH2CH2F), 3.62 (q, 2H, J ¼ 6.0 Hz, (CO)NHCH2CH2N), 4.58
(td, 2H, 2JHeF ¼ 47.7 Hz, J ¼ 5.0 Hz, CH2F), 7.81 (d, 1H, J ¼ 8.8 Hz, H-
8), 8.05 (dd, 1H, J ¼ 1.8, 8.8 Hz, H-7), 8.45 (br s, 1H, NH), 8.58 (d, 1H,
J ¼ 1.8 Hz, H-5), 9.62 (s, 1H, H-3); 13C NMR (100 MHz, CDCl3)
d 11.8,
37.3, 48.5, 52.9, 53.6 (d, 2JCeF ¼ 20 Hz), 82.5 (d, 1JCeF ¼ 167 Hz), 98.0,
v/v) 0.20; mp 60e62 ꢁC; IR (ATR diamond accessory)
1099, 1255, 1357, 1387, 1473, 1519, 1594, 1660, 1684, 2855, 2930,
2955, 3376 cmꢀ1; 1H NMR (200 MHz, CDCl3)
0.00 (s, 6H, (CH3)2Si),
n 775, 836,
130.9, 138.6, 139.6, 139.7, 144.0, 144.4, 144.6, 163.1; 19F NMR (CDCl3)
d
ꢀ220.22 (tt, 3JHeF ¼ 26.4 Hz, 2JHeF ¼ 47.4 Hz); MS m/z 416 (Mþ, 1),
d
104 (100), 76 (12), 56 (8).
0.84 (s, 9H, (CH3)3C), 1.07 (t, 3H, J ¼ 7.1 Hz, CH2CH3), 1.69 (quint., 2H,
J ¼ 6.7 Hz, NCH2CH2CH2O), 2.62 (m, 4H, CH2CH3, NCH2CH2CH2O),
2.70 (t, 2H, J ¼ 6.0 Hz, (CO)2NCH2CH2N), 3.56 (q, 2H, J ¼ 5.8 Hz,
(CO)2NCH2CH2N), 3.70 (t, 2H, J ¼ 6.2 Hz, NCH2CH2CH2O), 7.82 (d,
1H, J ¼ 8.8 Hz, H-8), 8.06 (dd, 1H, J ¼ 1.9, 8.8 Hz, H-7), 8.41 (br s, 1H,
NH), 8.60 (d, 1H, J ¼ 1.9 Hz, H-5), 9.64 (s, 1H, H-3); 13C NMR
4.2.4. N-[2-[N-[3-(tert-Butyldimethylsilyloxy)propyl]-N-
ethylamino]ethyl]phthalimide (10)
To a solution of phthalimide 9 [37] (1.00 g, 3.95 mmol) in
anhydrous acetonitrile (30 mL) were successively added, under
argon, potassium carbonate (1.09 g, 7.90 mmol) and commercially
available (3-bromopropoxy)-tert-butyldimethylsilane (1.00 g,
3.95 mmol). The mixture was then stirred at 50 ꢁC for 36 h. After
cooling to room temperature, a saturated aqueous sodium car-
bonate solution (90 mL) was added and the aqueous layer was then
extracted with dichloromethane (3 ꢃ 60 mL). The organic layers
were combined, dried on magnesium sulphate, filtered and evap-
orated under vacuum. The obtained liquid residue was purified by
chromatography (SiO2, CH2Cl2/EtOH, 98/2, v/v) to give compound
10 (600 mg, 1.54 mmol) as a colourless liquid. Yield 39%; Rf (SiO2,
(50 MHz, CDCl3)
d
ꢀ5.2 (2C), 12.1, 18.3, 26.0 (3C), 30.6, 37.3, 47.6,
49.9, 52.3, 61.1, 97.9, 130.9, 138.6, 139.6 (2C), 144.2, 144.4, 144.7,
162.9; ESI-MS m/z 543.2 [M þ H]þ.
4.2.7. N-[2-[N-Ethyl-N-(3-hydroxypropyl)amino]ethyl]-6-
iodoquinoxaline-2-carboxamide (13)
To a stirred solution of compound 12 (428 mg, 0.79 mmol) in
THF (13 mL) was added a solution of tetrabutylammonium fluoride
(TBAF) 1 M in THF (1.18 mL, 1.18 mmol). The mixture was stirred at
room temperature for 1.5 h and the reaction was stopped by
addition of a saturated aqueous sodium hydrogencarbonate solu-
tion (100 mL), followed by distilled water (50 mL) and then ethyl
acetate (50 mL). After decantation, the aqueous layer was extracted
with ethyl acetate (3 ꢃ 50 mL). The organic layers were combined,
washed with brine (50 mL), dried on magnesium sulphate, filtered
and evaporated under vacuum. The residue was then purified by
chromatography (SiO2, CH2Cl2/EtOH, 85/15 and then 80/20, v/v) to
give compound 13 (249 mg, 0.58 mmol) as a yellow solid. Yield 74%;
Rf (SiO2, CH2Cl2/EtOH, 9/1, v/v) 0.24; mp 78e80 ꢁC; IR (ATR dia-
CH2Cl2/EtOH, 98/2, v/v) 0.25; IR (ATR diamond accessory)
838, 1101, 1256, 1397, 1435, 1470, 1718, 2857, 2930, 2955 cmꢀ1
NMR (200 MHz, CDCl3)
n
777,
1H
ꢀ0.02 (s, 6H, (CH3)2Si), 0.84 (s, 9H,
;
d
(CH3)3C), 0.97 (t, 3H, J ¼ 7.1 Hz, CH2CH3), 1.57 (quint., 2H, J ¼ 6.8 Hz,
NCH2CH2CH2O), 2.53 (m, 4H, CH2CH3, NCH2CH2CH2O), 2.68 (t, 2H,
J ¼ 6.9 Hz, (CO)2NCH2CH2N), 3.53 (t, 2H, J ¼ 6.3 Hz, NCH2CH2CH2O),
3.75 (t, 2H, J ¼ 6.9 Hz, (CO)2NCH2CH2N), 7.69 (m, 2H, H-5, H-6), 7.82
(m, 2H, H-4, H-7); 13C NMR (50 MHz, CDCl3)
d
ꢀ5.3 (2C), 12.0, 18.3,
26.0 (3C), 30.5, 36.2, 47.6, 50.2, 50.9, 61.2, 123.2 (2C), 132.3 (2C),
133.9 (2C), 168.4 (2C); ESI-MS m/z 391.3 [M þ H]þ.
mond accessory)
n
1064, 1351, 1476, 1527, 1593, 1679, 2835, 2940,
1.04 (t, 3H, J ¼ 7.1 Hz,
3326, 3462 cmꢀ1; 1H NMR (200 MHz, CDCl3)
d
4.2.5. N-[3-(tert-Butyldimethylsilyloxy)propyl]-N-ethyl-1,2-
ethylenediamine (11)
CH3), 1.73 (quint., 2H, J ¼ 5.7 Hz, NCH2CH2CH2O), 2.64 (q, 2H,
J ¼ 7.1 Hz, CH2CH3), 2.74 (m, 4H, CONHCH2CH2N, NCH2CH2CH2O),
3.64 (q, 2H, J ¼ 6.1 Hz, CONHCH2CH2N), 3.82 (t, 2H, J ¼ 5.4 Hz,
NCH2CH2CH2O), 7.81 (d, 1H, J ¼ 8.8 Hz, H-8), 8.04 (dd, 1H, J ¼ 1.8,
8.8 Hz, H-7), 8.32 (br s, 1H, NH), 8.57 (d, 1H, J ¼ 1.8 Hz, H-5), 9.60 (s,
To a solution of phthalimide 10 (400 mg, 1.02 mmol) in absolute
ethanol (12 mL) was added hydrazine monohydrate (500
mL,
10.24 mmol). The mixture was stirred at room temperature for 18 h
and cooled to 0 ꢁC for 2 h. The resulting white precipitate was
filtered, washed successively with ice-cold ethanol (10 mL) and
dichloromethane (10 mL) and the filtrate was evaporated under
1H, H-3); 13C NMR (50 MHz, CDCl3)
d 11.6, 28.4, 37.5, 47.7, 52.8, 53.5,
63.6, 98.2, 130.9, 138.5, 139.5, 139.8, 143.8, 144.4, 144.5, 163.2; ESI-
MS m/z 429.1 [M þ H]þ.

848
A. Maisonial et al. / European Journal of Medicinal Chemistry 63 (2013) 840e853
4.2.8. N-[2-[N-Ethyl-N-(3-fluoropropyl)amino]ethyl]-6-
iodoquinoxaline-2-carboxamide (14)
(1.57 mL,1.57 mmol) under argon. The mixture was refluxed for 2 h.
After cooling to room temperature, the solvent was evaporated
under reduced pressure and the residue was purified by chroma-
tography (SiO2, CH2Cl2) to give compound 19 (174 mg, 0.72 mmol)
as an orange-coloured oil. Yield 56%; Rf (SiO2, CH2Cl2) 0.71; IR (CCl4)
To a solution of alcohol 13 (20 mg, 46.7 mmol) in anhydrous DME
(0.8 mL) was added under argon and at ꢀ50 ꢁC, DAST (12.5
mL,
93.4
m
mol). The reaction media was stirred at ꢀ50 ꢁC for 15 min,
then at room temperature for 1.5 h, and poured into a saturated
aqueous sodium hydrogencarbonate solution (10 mL). The mixture
was extracted with ethyl acetate (3 ꢃ 10 mL). The organic layers
were combined, dried on magnesium sulphate, filtered and evap-
orated under vacuum. The residue was purified by chromatography
n d 2.46 (s,
1179, 1181, 1381, 2950 cmꢀ1; 1H NMR (200 MHz, CDCl3)
3H, CH3), 4.74 (td, 2H, 5JHeF ¼ 6.7 Hz, J ¼ 1.7 Hz, CH2OTos), 4.87 (td,
2H, 2JHeF ¼ 33.7 Hz, J ¼ 1.7 Hz, CH2F), 7.36 (d, 2H, J ¼ 8.3 Hz, 2Har),
7.82 (d, 2H, J ¼ 8.3 Hz, 2Har); 13C NMR (50 MHz, acetone-d6)
d 21.5,
58.5 (d, 4JCeF ¼ 3 Hz), 70.9 (d, 1JCeF ¼ 164 Hz), 82.8 (d, 3JCeF ¼ 12 Hz),
(SiO2, EtOAc) to give compound 14 (7 mg, 16.3
m
mol) as a yellow oil.
1.10 (t,
83.8 (d, 2JCeF ¼ 22 Hz), 128.9 (2C), 130.9 (2C), 134.1, 146.3; 19F NMR
Yield 35%; Rf (SiO2, EtOAc) 0.36; 1H NMR (200 MHz, CDCl3)
d
(CDCl3)
d
ꢀ217.56.
3H, J ¼ 7.1 Hz, CH3), 1.82 (quint., 2H, J ¼ 6.5 Hz, NCH2CH2CH2F), 2.64
(m, 6H, CH2CH3, CONHCH2CH2N, NCH2CH2CH2F), 3.57 (q, 2H,
J ¼ 5.9 Hz, CONHCH2CH2N), 3.89 (m, 2H, NCH2CH2CH2F), 7.87 (d,
1H, J ¼ 8.8 Hz, H-8), 8.08 (dd, 1H, J ¼ 1.9, 8.8 Hz, H-7), 8.41 (br s, 1H,
NH), 8.60 (d, 1H, J ¼ 1.9 Hz, H-5), 9.63 (s, 1H, H-3); 19F NMR (CD3CN)
4.2.12. N-[2-[[N-Ethyl-N-(4-fluorobut-2-ynyl)]amino]ethyl]-6-
iodoquinoxaline-2-carboxamide (20)
To a stirred solution of compound 19 (491 mg, 2.03 mmol) in
anhydrous acetonitrile (17 mL) was added dropwise amine 15 [36]
(0.50 g, 1.35 mmol). After 72 h at room temperature, water (5 mL)
and a 2.0 M aqueous sodium carbonate solution (5 mL) were added.
The resulting solution was extracted with dichloromethane
(3 ꢃ 10 mL). The organic layers were combined, dried on magne-
sium sulphate, filtered and evaporated under vacuum. The residue
was purified by chromatography (Al2O3, CH2Cl2/EtOH, 99/1, v/v) to
give compound 20 (226 mg, 0.51 mmol) as a yellow oil. Yield 38%; Rf
d
ꢀ220.47; ESI-MS m/z 431.1 [M þ H]þ.
4.2.9. N-[2-[N-Ethyl-N-[(E)-4-fluorobut-2-enyl]amino]ethyl]-6-
iodoquinoxaline-2-carboxamide (17)
To a solution of (E)-4-fluoro-but-2-enyl toluene-4-sulfonate (16)
[38] (380 mg, 1.56 mmol) in anhydrous acetonitrile (16 mL) was
added dropwise and under argon
a
solution of N-(2-
ethylaminoethyl)-6-iodoquinoxaline-2-carboxamide (15) [36]
(464 mg, 1.13 mmol) in anhydrous acetonitrile (17 mL). The
mixture was stirred at room temperature for 60 h. The solvent was
then evaporated under vacuum and the residue was purified by
chromatography (Al2O3, CH2Cl2/EtOH, 99/1, v/v) to give compound
17 (235 mg, 0.53 mmol) as a yellow solid. Yield 47%; Rf (Al2O3,
(Al2O3, CH2Cl2/EtOH, 99/1, v/v) 0.86; IR (CCl4)
n
1475, 1522, 1685,
2928 cmꢀ1; 1H NMR (200 MHz, acetone-d6)
d
1.11 (t, 3H, J ¼ 7.2 Hz,
CH3), 2.64 (q, 2H, J ¼ 7.2 Hz, CH2CH3), 2.80 (t, 2H, J ¼ 6.1 Hz, (CO)
NHCH2CH2N), 3.59 (m, 4H, (CO)NHCH2CH2N, NCH2C^C), 4.97 (td,
2H, 2JHeF ¼ 47.5 Hz, J ¼ 1.7 Hz, CH2F), 7.80 (d, 1H, J ¼ 8.8 Hz, H-8),
8.05 (dd, 1H, J ¼ 1.8, 8.8 Hz, H-7), 8.25 (br s, 1H, NH), 8.58 (d, 1H,
J ¼ 1.8 Hz, H-5), 9.62 (s, 1H, H-3); 13C NMR (50 MHz, acetone-d6)
CH2Cl2/EtOH, 99/1, v/v) 0.36; mp 50e52 ꢁC (dec.); IR (CCl4)
1353, 1475, 1520, 1682, 2817, 2850e3000, 3409 cmꢀ1 1H NMR
(200 MHz, CDCl3)
1.08 (t, 3H, J ¼ 7.1 Hz, CH3), 2.63 (q, 2H,
J ¼ 7.1 Hz, CH2CH3), 2.72 (t, 2H, J ¼ 6.2 Hz, (CO)NHCH2CH2N), 3.21
n 1160,
;
d
12.9, 37.7, 42.2 (d, 4JCeF ¼ 3 Hz), 48.3, 52.9, 71.5 (d, 1JCeF ¼ 161 Hz),
2
3
d
80.3 (d, JCeF ¼ 22 Hz), 85.5 (d, JCeF ¼ 12 Hz), 98.3, 131.8, 139.1,
140.2, 140.5, 145.0, 145.3, 145.5, 163.5; 19F NMR (CDCl3)
d
ꢀ213.79;
(m, 2H, NCH2CH]CHCH2F), 3.57 (q, 2H,
J
¼
5.9 Hz, (CO)
ESI-MS m/z 441.1 [M þ H]þ.
NHCH2CH2N), 4.81 (dd, 2H, 2JHeF ¼ 47.0 Hz, J ¼ 4.3 Hz, CH2F), 5.90
(m, 2H, NCH2CH]CHCH2F), 7.81 (d, 1H, J ¼ 8.8 Hz, H-8), 8.07 (dd,
1H, J ¼ 1.8, 8.8 Hz, H-7), 8.32 (m, 1H, NH), 8.59 (d, 1H, J ¼ 1.8 Hz, H-
4.2.13. 2,2,3,3-Tetramethyl-4,7,10,13-tetraoxa-3-silapentadecan-
15-ol (21)
A solution of sodium hydride (60% in mineral oil, 2.94 g,
73.5 mmol) in anhydrous THF (135 mL) was stirred at room tem-
5), 9.63 (s, 1H, H-3); 13C NMR (50 MHz, CDCl3)
d
12.2, 37.4, 47.8, 51.9,
4
2
55.2 (d, JCeF ¼ 1 Hz), 83.0 (d, 1JCeF ¼ 162 Hz), 98.1, 127.4 (d, JCe
¼ 17 Hz), 130.9, 133.2 (d, 3JCeF ¼ 12 Hz), 138.7, 139.7, 139.8, 144.1,
perature for 5 min under argon. Dried tetraethylene glycol
F
144.5, 144.7, 163.0; 19F NMR (CDCl3)
d
ꢀ211.13 (t, 2JHeF ¼ 48.2 Hz);
(8.80 mL, 51.0 mmol) was added dropwise at 0 ꢁC and the reaction
mixture was stirred for 5 more min before addition of tert-butyl-
dimethylsilyl chloride (TBDMSCl, 12.00 g, 79.6 mmol). The resulting
mixture was stirred at room temperature for 1 h and the reaction
was stopped by addition of water (150 mL). The mixture was
extracted with ethyl acetate (3 ꢃ 100 mL). The organic layers were
combined, washed successively with a saturated aqueous ammo-
nium chloride solution (150 mL) and brine (150 mL), dried on
magnesium sulphate, filtered and evaporated under vacuum. The
crude product was purified by chromatography (SiO2 pad, EtOAc/
pentane, 75/25, v/v then 50/50, v/v and finally CH2Cl2/EtOH, 98/2,
v/v) to yield compound 21 (6.42 g, 20.8 mmol) as a yellow oil. Yield
41%; Rf (SiO2, EtOAc/pentane, 75/25, v/v) 0.16; IR (ATR diamond
ESI-MS m/z 442.9 [M þ H]þ.
4.2.10. 1,4-bis(p-Toluenesulfonyloxy)but-2-yne (18)
This compound was synthesized according to the procedure
described by Shine et al. for the tosylation of hex-3-yne-2,4-diol [39].
Briefly, to a stirred solution of p-toluenesulfonyl chloride (28.2 g,
0.15 mol) in diethylether (340 mL) was added commercially available
1,4-butynediol (5.00 g, 58.1 mmol). The mixture was cooledto ꢀ15 ꢁC
and potassium hydroxide (23.7 g, 0.42 mmol) was added dropwise.
The resulting solution was stirred at 0 ꢁC for 2 h and poured into cold
water (200 mL). After return back to room temperature, the solution
was decanted, the organic layer was collected while the aqueous
layer was extracted with dichloromethane (3 ꢃ 200 mL). The organic
layers were combined, dried on magnesium sulphate, filtered and
evaporated under vacuum to give compound 18 (21.8 g, 55.3 mmol)
as a beige solid. Yield 95%; mp 94e96 ꢁC (Lit. [45]. : 94e95 ꢁC); IR
accessory)
3600 cmꢀ1; 1H NMR (200 MHz, CDCl3)
(s, 9H, (CH3)3C), 2.63 (br s, 1H, OH), 3.65 (m, 16H, 8CH2O); 13C NMR
(50 MHz, CDCl3)
n
778, 837, 1109, 1255, 1472, 2859, 2930, 3300e
d
0.05 (s, 6H, (CH3)2Si), 0.88
d
ꢀ5.2 (2C), 18.4, 26.0 (3C), 61.8, 62.8, 70.4e70.7
(KBr)
n
934, 1178, 1350, 1374, 2991 cmꢀ1; 1H NMR (200 MHz, CDCl3)
(4C), 72.6, 72.7; MS m/z 251 (M-57, 9), 163 (26), 119 (46), 103 (55),
d
2.45 (s, 6H, 2CH3), 4.57 (s, 4H, 2CH2OTos), 7.35 (d, 4H, J ¼ 8.3 Hz,
89 (100), 75 (93).
4Har), 7.76 (d, 4H, J ¼ 8.3 Hz, 4Har).
4.2.14. 2,2,3,3-Tetramethyl-4,7,10,13,16,19,22,25-octaoxa-3-
silaheptacosan-27-ol (22)
4.2.11. 4-Fluoro-but-2-ynyl toluene-4-sulfonate (19)
To a stirred solution of ditosyl 18 (503 mg, 1.28 mmol) in
anhydrous THF (20 mL) was added, a solution of TBAF 1.0 M in THF
This compound was synthesized according to the procedure
described for 21, starting from octaethylene glycol (6.01 g,

A. Maisonial et al. / European Journal of Medicinal Chemistry 63 (2013) 840e853
849
16.2 mmol), sodium hydride (60% in mineral oil, 0.91 g, 22.7 mmol)
and TBDMSCl (3.67 g, 24.3 mmol). Reaction time: 4 h at room
temperature; the purification was performed using column chro-
matography (SiO2, EtOAc/EtOH, 95/5, v/v then 8/2, v/v) to give
compound 22 (5.32 g, 11.0 mmol) as a yellow oil. Yield 68%; Rf (SiO2,
layers were combined, dried on magnesium sulphate, filtered and
evaporated under vacuum. The oily residue was purified by chro-
matography (SiO2, EtOAc) to afford compound 25 (4.38 g,
8.61 mmol) as a pale yellow oil. Yield 72%; Rf (SiO2, EtOAc) 0.25; IR
(NaCl)
n ;
837, 1105, 1254, 1396, 1468, 1592, 1713, 2858, 2900 cmꢀ1
EtOAc/EtOH, 95/5, v/v) 0.21; IR (NaCl)
2927, 3100e3600 cmꢀ1 1H NMR (200 MHz, CDCl3)
(CH3)2Si), 0.83 (s, 9H, (CH3)3C), 3.58 (m, 32H, 16CH2O); 13C NMR
(50 MHz, CDCl3)
n
778, 836, 1106, 1254, 2873,
1H NMR (200 MHz, CDCl3)
d 0.04 (s, 6H, (CH3)2Si), 0.87 (s, 9H,
;
d
0.00 (s, 6H,
(CH3)3C), 0.97 (t, 3H, J ¼ 7.1 Hz, CH2CH3), 2.61 (q, 2H, J ¼ 7.1 Hz,
CH2CH3), 2.74 (m, 4H, (CO)2NCH2CH2N, NCH2CH2O), 3.57 (m, 12H,
6CH2O), 3.76 (m, 4H, CH2OSi, (CO)2NCH2CH2N), 7.70 (m, 2H, H-5, H-
d
ꢀ5.2 (2C), 18.4, 26.0 (3C), 61.7, 62.7, 70.4e70.6
(12C), 72.6, 72.7; MS m/z 251 (13), 233 (6), 207 (53), 189 (12), 163
6), 7.80 (m, 2H, H-4, H-7); 13C NMR (50 MHz, CDCl3)
d
ꢀ5.2 (2C),
(34), 145 (15), 119 (49), 115 (27), 89 (100), 75 (46).
12.0, 18.4, 26.0 (3C), 36.2, 48.4, 51.5, 53.0, 62.8, 69.8, 70.5, 70.6, 70.7,
70.8, 72.7, 123.2 (2C), 132.3 (2C), 133.9 (2C), 168.4 (2C); ESI-MS m/z
509.4 [M þ H]þ.
4.2.15. 15-Iodo-2,2,3,3-tetramethyl-4,7,10,13-tetraoxa-3-
silapentadecane (23)
To a stirred solution of alcohol 21 (5.00 g, 16.2 mmol) in anhy-
drous dichloromethane (150 mL) were successively added imid-
azole (1.44 g, 21.1 mmol), triphenylphosphine (5.53 g, 21.1 mmol)
and iodine (5.35 g, 21.1 mmol) at 0 ꢁC under argon. The mixture was
stirred at 0 ꢁC for 30 min, then at room temperature for 1 h. A 10%
aqueous sodium bisulfite solution (170 mL) was added and the
mixture was forcefully stirred for 2 min (until fading). After
decantation the organic layer was collected and the aqueous layer
was extracted with dichloromethane (3 ꢃ 80 mL). The organic
layers were combined, dried on magnesium sulphate, filtered and
evaporated under vacuum. The obtained solid was suspended in n-
pentane (150 mL) and the mixture was stirred at room temperature
for 45 min before filtration. The precipitate was washed with n-
pentane (30 mL). The filtrate was evaporated under vacuum and the
oily residue was purified by chromatography (SiO2 pad, CH2Cl2/
EtOH, 98/2, v/v) to give compound 23 as a pale yellow oil (5.62 g,
13.4 mmol). Yield 83%; Rf (SiO2, CH2Cl2/EtOH, 98/2, v/v) 0.25; IR
4.2.18. N-(28-Ethyl-2,2,3,3-tetramethyl-4,7,10,13,16,19,22,25-
octaoxa-28-aza-3-silaheptacosan-30-yl)phthalimide (26)
To a stirred solution of phthalimide 9 [37] (489 mg, 1.92 mmol)
in anhydrous acetonitrile (15 mL) was added potassium carbonate
(530 mg, 3.84 mmol) under argon. The reaction mixture was stirred
at room temperature for 1 h before addition of compound 24
(1.14 g, 1.92 mmol). The mixture was heated at 50 ꢁC for 7 d, cooled
to room temperature and partitioned between a saturated aqueous
sodium carbonate solution (50 mL) and dichloromethane (30 mL).
After decantation, the organic layer was collected while the
aqueous layer was extracted with dichloromethane (3 ꢃ 30 mL).
The organic layers were combined, dried on magnesium sulphate,
filtered and evaporated under vacuum. The oily residue was puri-
fied by chromatography (SiO2, CH2Cl2/EtOH, 95/5, v/v) to afford
compound 26 (854 mg,1.25 mmol) as a pale yellow oil. Yield 65%; Rf
(SiO2, CH2Cl2/EtOH, 95/05, v/v) 0.32; IR (ATR diamond accessory)
838, 1112, 1255, 1397, 1436, 1469, 1717, 2862, 2929 cmꢀ1 1H NMR
(200 MHz, CDCl3) 0.05 (s, 6H, (CH3)2Si), 0.88 (s, 9H, (CH3)3C), 0.99
n
;
(ATR diamond accessory)
n
778, 837, 1109, 1256, 1472, 2858,
0.04 (s, 6H, (CH3)2Si), 0.87
d
2928 cmꢀ1; 1H NMR (200 MHz, CDCl3)
d
(t, 3H, J ¼ 7.1 Hz, CH2CH3), 2.66 (q, 2H, J ¼ 7.5 Hz, CH2CH3), 2.73 (m,
(s, 9H, (CH3)3C), 3.24 (t, 2H, J ¼ 7.0 Hz, CH2I), 3.53 (t, 2H, J ¼ 5.4 Hz,
4H, (CO)2NCH2CH2N, NCH2CH2O), 3.56 (m, 4H, NCH2CH2O, OCH2
-
OCH2CH2OSi), 3.64 (m, 8H, 4CH2O), 3.73 (m, 4H, ICH2CH2, CH2OSi);
CH2OSi), 3.63 (m, 24H, 12CH2O), 3.76 (m, 4H, (CO)2NCH2CH2N,
13C NMR (50 MHz, CDCl3)
70.3e70.8 (4C), 72.1, 72.7; MS m/z 361 (M-57, 3), 317 (14), 273 (4),
d
ꢀ5.2 (2C), 3.0, 18.4, 26.0 (3C), 62.8,
CH2OSi), 7.70 (m, 2H, H-5, H-6), 7.83 (m, 2H, H-4, H-7); 13C NMR
(50 MHz, CDCl3)
d
ꢀ5.2 (2C), 12.0, 18.4, 26.0 (3C), 36.1, 48.5, 51.4,
229 (5), 199 (13), 155 (100).
53.0, 62.8, 70.6 (13C), 72.7, 123.2 (2C), 132.3 (2C), 133.9 (2C), 168.4
(2C); ESI-MS m/z 685.5 [M þ H]þ.
4.2.16. 27-Iodo-2,2,3,3-tetramethyl-4,7,10,13,16,19,22,25-octaoxa-
3-silaheptacosane (24)
4.2.19. 16-Ethyl-2,2,3,3-tetramethyl-4,7,10,13-tetraoxa-16-aza-3-
silaoctadecan-18-amine (27)
This compound was synthesized according to the procedure
described for 23, starting from compound 22 (2.00 g, 4.13 mmol),
imidazole (365 mg, 5.36 mmol), triphenylphosphine (1.41 g,
5.36 mmol) and iodine (1.36 g, 5.36 mmol). Reaction time: 2 d at
room temperature; the purification was performed using column
chromatography (SiO2, EtOAc) to give compound 24 as a pale yel-
low oil (1.89 g, 3.18 mmol). Yield 77%; Rf (SiO2, EtOAc) 0.30; IR
To a stirred solution of 25 (1.50 g, 2.95 mmol) in ethanol
(150 mL) was added hydrazine monohydrate (1.43 mL, 29.5 mmol).
The mixture was stirred at room temperature for 20 h and then
cooled at 0 ꢁC for 3 h. The white precipitate was filtered and washed
with ice-cold ethanol (50 mL) and dichloromethane (50 mL) and
the filtrate was evaporated under vacuum. The residue was sus-
pended in dichloromethane (20 mL) and the remaining precipitate
was filtered again and washed with dichloromethane (20 mL). The
last steps were repeated until no more white solid appeared after
evaporation of the filtrate. Amine 27 (1.12 g, 2.95 mmol) was ob-
tained as a pale yellow oil and used without further purification.
Yield quant.; Rf (Al2O3, CH2Cl2/EtOH, 8/2, v/v) 0.32; IR (ATR dia-
(NaCl)
CDCl3)
n
778, 835, 1108, 1253, 2859, 2927 cmꢀ1; 1H NMR (200 MHz,
0.06 (s, 6H, (CH3)2Si), 0.89 (s, 9H, (CH3)3C), 3.26 (t, 2H,
d
J ¼ 7.0 Hz, CH2I), 3.55 (t, 2H, J ¼ 5.5 Hz, OCH2CH2OSi), 3.65 (m, 24H,
12CH2O), 3.70 (m, 4H, CH2CH2I, OCH2CH2OSi); 13C NMR (50 MHz,
CDCl3)
d
ꢀ5.2 (2C), 3.0, 18.4, 26.0 (3C), 62.8, 70.3e70.7 (12C), 72.1,
72.7; MS m/z 361 (2), 331 (1), 287 (6), 243 (8), 233 (4), 199 (28), 189
(8), 159 (16), 155 (100), 145 (9), 115 (15).
mond accessory)
n
834, 1107, 1252, 1472, 1574, 2857, 2929, 3100e
0.05 (s, 6H, (CH3)2Si), 0.88
3300 cmꢀ1; 1H NMR (200 MHz, CDCl3)
d
4.2.17. N-(16-Ethyl-2,2,3,3-tetramethyl-4,7,10,13-tetraoxa-16-aza-
3-silaoctadecan-18-yl)phthalimide (25)
(s, 9H, (CH3)3C), 1.01 (t, 3H, J ¼ 7.1 Hz, CH2CH3), 2.68 (m, 8H,
CH2CH3, H2NCH2CH2N, H2NCH2CH2N, NCH2CH2O), 3.55 (m, 12H,
To a stirred solution of phthalimide 9 [37] (3.04 g, 12.0 mmol) in
anhydrous acetonitrile (80 mL) were successively added potassium
carbonate (1.65 g, 12.0 mmol) and compound 23 (5.00 g,
12.0 mmol) under argon. The mixture was heated at 50 ꢁC for 6 d.
After cooling to room temperature, a saturated aqueous sodium
carbonate solution was added (200 mL) and the resulting solution
was extracted with dichloromethane (3 ꢃ 150 mL). The organic
6CH2O), 3.72 (m, 2H, CH2OSi); 13C NMR (50 MHz, CDCl3)
11.9, 18.4, 26.0 (3C), 39.7, 48.8, 52.7, 56.4, 62.8, 70.5 (5C), 72.7.
d
ꢀ5.2 (2C),
4.2.20. 28-Ethyl-2,2,3,3-tetramethyl-4,7,10,13,16,19,22,25-octaoxa-
28-aza-3-silaheptacosan-30-amine (28)
This compound was synthesized according to the procedure
described for 27, starting from compound 26 (607 mg, 0.89 mmol)

850
A. Maisonial et al. / European Journal of Medicinal Chemistry 63 (2013) 840e853
and hydrazine monohydrate (430
mL, 8.86 mmol). Reaction time:
2 h. The reaction was stopped by addition of a saturated aqueous
20 h at room temperature to give amine 28 (492 mg, 0.89 mmol) as
a pale yellow oil which was used without further purification. Yield
sodium hydrogencarbonate solution (80 mL) followed by distilled
water (40 mL) and then ethyl acetate (40 mL). After decantation, the
aqueous layer was extracted with ethyl acetate (3 ꢃ 40 mL). The
organic layers were combined, washed with brine (40 mL), dried on
magnesium sulphate, filtered and evaporated under vacuum. The
obtained residue was purified by chromatography (SiO2, CH2Cl2/
EtOH, 95/5 to 80/20, v/v) to give compound 31 (132 mg, 0.24 mmol)
as a pale yellow oil. Yield 93%; Rf (SiO2, CH2Cl2/EtOH, 80/20, v/v)
quant.; Rf (Al2O3, CH2Cl2/EtOH, 9/1, v/v) 0.23; IR (NaCl)
1252, 1299, 1472, 1648, 2859, 2929, 3100e3600 cmꢀ1
(200 MHz, CDCl3) 0.04 (s, 6H, (CH3)2Si), 0.87 (s, 9H, (CH3)3C), 1.00
n
837, 1108,
;
1H NMR
d
(t, 3H, J ¼ 7.1 Hz, CH2CH3), 2.61 (m, 8H, CH2CH3, H2NCH2CH2N,
H2NCH2CH2N, NCH2CH2O), 3.51 (t, 2H, J ¼ 6.3 Hz, NCH2CH2O), 3.53
(t, 2H, J ¼ 5.4 Hz, OCH2CH2OSi), 3.63 (m, 24H, 12CH2O), 3.74 (t, 2H,
J ¼ 5.4 Hz, CH2OSi); 13C NMR (50 MHz, CDCl3)
d
ꢀ5.2 (2C), 12.0, 18.4,
0.26; IR (NaCl)
n 1125, 1353, 1473, 1527, 1592, 1671, 2871, 3300e
26.0 (3C), 39.9, 48.7, 53.0, 56.9, 62.8, 70.1e70.6 (13C), 72.7.
3400 cmꢀ1; 1H NMR (200 MHz, CDCl3)
d
1.10 (t, 3H, J ¼ 7.1 Hz, CH3),
2.82 (m, 6H, CH2CH3, NCH2CH2O, CONHCH2CH2N), 3.60 (m, 16H,
CONHCH2CH2N, 7CH2O), 7.83 (d, 1H, J ¼ 8.8 Hz, H-8), 8.06 (dd, 1H,
J ¼ 1.8, 8.8 Hz, H-7), 8.57 (br s, 1H, NH), 8.59 (d, 1H, J ¼ 1.8 Hz, H-5),
4.2.21. N-(16-Ethyl-2,2,3,3-tetramethyl-4,7,10,13-tetraoxa-16-aza-
3-silaoctadecan-18-yl)-6-iodoquinoxaline-2-carboxamide (29)
To a stirred suspension of activated ester 7 [36] (305 mg,
0.72 mmol) in anhydrous THF (7 mL) was added under argon a
solution of 27 (430 mg, 1.14 mmol) in anhydrous THF (8 mL). The
mixture was stirred at room temperature for 21 h and the solvent
was then evaporated under vacuum. The obtained residue was
dissolved in dichloromethane (15 mL) and a 1 N aqueous sodium
hydroxide solution was added (45 mL). After decantation, the
organic layer was collected while the aqueous layer was extracted
with dichloromethane (3 ꢃ 15 mL). The organic layers were com-
bined, washed with a 5% aqueous sodium carbonate solution
(2 ꢃ 45 mL), dried on magnesium sulphate, filtered and evaporated
under vacuum. The oily residue was purified by chromatography
(SiO2, CH2Cl2/EtOH, 9/1, v/v) to provide compound 29 (459 mg,
0.70 mmol) as a pale yellow oil. Yield 96%; Rf (SiO2, CH2Cl2/EtOH, 9/
9.62 (s, 1H, H-3); 13C NMR (50 MHz, CDCl3)
d 11.7, 37.3, 48.9, 52.9,
53.1, 61.7, 69.7, 70.3e70.6 (4C), 72.7, 98.0, 130.9, 138.6, 139.7 (2C),
144.2, 144.4, 144.7, 163.2; ESI-MS m/z 547.2 [M þ H]þ.
4.2.24. N-(24-Ethyl-1-hydroxy-3,6,9,12,15,18,21-heptaoxa-24-
azahexacosan-26-yl)-6-iodoquinoxaline-2-carboxamide (32)
This compound was synthesized according to the procedure
described for 31, starting from compound 30 (425 mg, 0.51 mmol)
and TBAF 1 M in THF (762 mL, 0.76 mmol). Reaction time: 1.5 h at
room temperature; the purification was performed using column
chromatography (SiO2, CH2Cl2/EtOH, 90/10 and then 80/20, v/v) to
afford compound 32 as a yellow oil (273 mg, 0.38 mmol). Yield 75%;
Rf (SiO2, CH2Cl2/EtOH, 80/20, v/v) 0.36; IR (ATR diamond accessory)
n
1127, 1353, 1474, 1529, 1593, 1675, 2800e3000, 3100e3600 cmꢀ1
;
1, v/v) 0.30; IR (NaCl)
1676, 1714, 2857, 2928, 3025, 3300e3400 cmꢀ1; 1H NMR (200 MHz,
CDCl3) 0.04 (s, 6H, (CH3)2Si), 0.87 (s, 9H, (CH3)3C), 1.08 (t, 3H,
7.1 Hz, CH2CH3), 2.78 (m, 6H, CH2CH3, NCH2CH2O,
n
834, 1108, 1253, 1353, 1387, 1472, 1523, 1592,
1H NMR (200 MHz, CDCl3)
d
1.06 (t, 3H, J ¼ 7.1 Hz, CH3), 2.66 (q, 2H,
J ¼ 7.1 Hz, CH2CH3), 2.76 (m, 4H, NCH2CH2O, CONHCH2CH2N), 2.91
(m, 1H, OH), 3.60 (m, 32H, CONHCH2CH2N, 15CH2O), 7.81 (d, 1H,
J ¼ 8.8 Hz, H-8), 8.05 (dd,1H, J ¼ 1.8, 8.8 Hz, H-7), 8.44 (br s,1H, NH),
8.58 (d, 1H, J ¼ 1.8 Hz, H-5), 9.61 (s, 1H, H-3); 13C NMR (50 MHz,
d
J
¼
CONHCH2CH2N), 3.60 (m, 16H, CONHCH2CH2N, 7CH2O), 7.82 (d, 1H,
J ¼ 8.8 Hz, H-8), 8.07 (dd,1H, J ¼ 1.8, 8.8 Hz, H-7), 8.46 (br s,1H, NH),
8.60 (d, 1H, J ¼ 1.8 Hz, H-5), 9.63 (s, 1H, H-3); 13C NMR (50 MHz,
CDCl3) d 12.1, 37.5, 48.7, 52.9, 53.0, 61.7, 70.0e70.6 (13C), 72.7, 98.0,
130.9, 138.6, 139.7 (2C), 144.2, 144.4, 144.7, 163.0; ESI-MS m/z 723.3
CDCl3)
d
ꢀ5.2 (2C), 12.1, 18.4, 26.0 (3C), 37.4, 48.7, 52.9, 53.0, 62.7,
[M þ H]þ.
70.7 (5C), 72.7, 98.0, 130.9, 138.6, 139.7 (2C), 144.2, 144.4, 144.7,
163.0; ESI-MS m/z 661.3 [M þ H]þ.
4.2.25. N-(12-Ethyl-1-fluoro-3,6,9-trioxa-12-azatetradecan-14-yl)-
6-iodoquinoxaline-2-carboxamide (33)
4.2.22. N-(28-Ethyl-2,2,3,3-tetramethyl-4,7,10,13,16,19,22,25-
octaoxa-28-aza-3-silaheptacosan-30-yl)-6-iodoquinoxaline-2-
carboxamide (30)
This compound was synthesized according to the procedure
described for 29, starting from compound 28 (450 mg, 0.81 mmol)
and activated ester 7 (220 mg, 0.52 mmol). Reaction time: 21 h at
room temperature; the purification was performed using column
chromatography (SiO2, CH2Cl2/EtOH, 95/5, v/v then 9/1, v/v) to
provide compound 30 (435 mg, 0.52 mmol) as a pale yellow oil.
This compound was synthesized according to the procedure
described for 14, starting from compound 31 (100 mg, 0.18 mmol)
and DAST (49
m
L, 0.37 mmol). Reaction time: 15 min at ꢀ50 ꢁC and
1.5 h at room temperature; the purification was performed using
column chromatography (SiO2, EtOAc/EtOH, 85/15, v/v, 0.5%
NH4OH) to yield compound 33 as a yellow oil (68 mg, 0.12 mmol).
Yield 68%; Rf (SiO2, EtOAc/EtOH, 85/15, v/v) 0.30; IR (ATR diamond
accessory)
n
1046, 1107, 1352, 1473, 1523, 1592, 1669, 2868, 2916,
3300e3400 cmꢀ1
;
1H NMR (200 MHz, CDCl3)
d
1.07 (t, 3H,
Yield 99%; Rf (SiO2, CH2Cl2/EtOH, 9/1, v/v) 0.28; IR (NaCl)
1252, 1352, 1473, 1527, 1592, 1677, 2860, 2927, 3300e3400 cmꢀ1
1H NMR (200 MHz, CDCl3)
0.03 (s, 6H, (CH3)2Si), 0.85 (s, 9H,
n
835,1107,
J ¼ 7.1 Hz, CH3), 2.65 (q, 2H, J ¼ 7.1 Hz, CH2CH3), 2.77 (m, 4H,
;
NCH2CH2O, CONHCH2CH2N), 3.61 (m,12H, CONHCH2CH2N, 5CH2O),
2
d
3.69 (m, 2H, CH2CH2F), 4.53 (td, 2H, JHeF ¼ 47.7 Hz, J ¼ 4.1 Hz,
(CH3)3C), 1.04 (t, 3H, J ¼ 7.1 Hz, CH2CH3), 2.65 (q, 2H, J ¼ 7.1 Hz,
CH2CH3), 2.74 (m, 4H, NCH2CH2O, CONHCH2CH2N), 3.61 (m, 32H,
CONHCH2CH2N, 15CH2O), 7.80 (d, 1H, J ¼ 8.8 Hz, H-8), 8.05 (dd, 1H,
J ¼ 1.8, 8.8 Hz, H-7), 8.41 (br s, 1H, NH), 8.57 (d, 1H, J ¼ 1.8 Hz, H-5),
CH2F), 7.81 (d, 1H, J ¼ 8.8 Hz, H-8), 8.07 (dd, 1H, J ¼ 1.8, 8.8 Hz, H-7),
8.42 (br s, 1H, NH), 8.60 (d, 1H, J ¼ 1.8 Hz, H-5), 9.63 (s, 1H, H-3); 13
C
NMR (50 MHz, CDCl3)
d 11.5, 37.1, 48.9, 52.8 (2C), 70.8 (6C), 83.2
(1JCeF ¼ 169 Hz), 98.1, 131.0, 138.6, 139.5, 139.7, 144.1, 144.4, 144.7,
9.61 (s, 1H, H-3); 13C NMR (50 MHz, CDCl3)
d
ꢀ5.2 (2C), 12.2, 18.4,
163.3; 19F NMR (CDCl3)
d
ꢀ223.1; ESI-MS m/z 549.2 [M þ H]þ.
26.0 (3C), 37.6, 48.7, 52.9, 53.0, 62.8, 70.2e70.6 (13C), 72.7, 97.9,
130.9, 138.6, 139.7 (2C), 144.2, 144.4, 144.7, 163.0; ESI-MS m/z 837.4
[M þ H]þ.
4.2.26. N-(24-Ethyl-1-fluoro-3,6,9,12,15,18,21-heptaoxa-24-
azahexacosan-26-yl)-6-iodoquinoxaline-2-carboxamide (34)
This compound was synthesized according to the procedure
described for 14, starting from compound 32 (94 mg, 0.13 mmol)
4.2.23. N-(12-Ethyl-1-hydroxy-3,6,9-trioxa-12-azatetradecan-14-
yl)-6-iodoquinoxaline-2-carboxamide (31)
and DAST (34.4
m
L, 0.26 mmol). Reaction time: 15 min at ꢀ50 ꢁC and
To a stirred solution of compound 29 (173 mg, 0.26 mmol) in
1.5 h at room temperature; the purification was performed using
THF (8 mL) was added a solution of TBAF 1 M in THF (393
mL,
column chromatography (SiO2, EtOAc/EtOH, 80/20, v/v, 0.5%
0.39 mmol). The mixture was then stirred at room temperature for
NH4OH) to yield compound 34 as a yellow oil (63 mg, 86.9 mmol).

A. Maisonial et al. / European Journal of Medicinal Chemistry 63 (2013) 840e853
851
Yield 67%; Rf (SiO2, EtOAc/EtOH, 5/5, v/v, 0.5% NH4OH) 0.32; IR (ATR
diamond accessory) 1129, 1353, 1474, 1527, 1593, 1675, 2800e
3000, 3300e3400 cmꢀ1 1H NMR (200 MHz, CDCl3)
1.07 (t, 3H,
¼ 14 Hz), 130.7, 138.4, 139.4, 139.6, 144.0, 144.2, 144.5, 145.3, 162.9;
F
n
19F NMR (CDCl3)
d
ꢀ223.21; ESI-MS m/z 498.0 [M þ H]þ.
;
d
J ¼ 7.1 Hz, CH3), 2.67 (q, 2H, J ¼ 7.1 Hz, CH2CH3), 2.79 (m, 4H,
NCH2CH2O, CONHCH2CH2N), 3.61 (m, 30H, CONHCH2CH2N,
14CH2O), 4.55 (td, 2H, 2JHeF ¼ 47.8 Hz, J ¼ 4.1 Hz, CH2F), 7.83 (d, 1H,
J ¼ 8.8 Hz, H-8), 8.08 (dd,1H, J ¼ 1.6, 8.8 Hz, H-7), 8.46 (br s,1H, NH),
8.61 (d, 1H, J ¼ 1.6 Hz, H-5), 9.64 (s, 1H, H-3); 19F NMR (CDCl3)
4.2.29. N-[2-[[N-Ethyl-N-(2-fluoroethyl)]amino]ethyl]-6-
iodoquinoxaline-2-carboxamide dihydrochloride salt (38)
To a stirred solution of compound 8 (180 mg, 0.43 mmol) in
anhydrous dichloromethane (5 mL) was added under argon an
anhydrous 2 N hydrochloric acid solution in anhydrous dieth-
ylether (10 mL). After 10 min, the solvent was evaporated under
vacuum. The residue was suspended in anhydrous diethylether
(10 mL) and the mixture was stirred under argon at room tem-
perature for 14 h. The precipitate was then filtered and dried under
reduce pressure to give dihydrochloride salt 38 (166 mg,
d
ꢀ223.2; ESI-MS m/z 725.2 [M þ H]þ; Anal. (C29H46O8IN4F, 3H2O)
calculated: C: 44.73; H: 6.73; N: 7.20; found: C: 44.84; H: 7.16, N:
5.82.
4.2.27. N-[2-[(N-Ethyl)-(N-prop-2-ynyl)amino]ethyl]-6-
iodoquinoxaline-2-carboxamide (35)
To a solution of compound 15 [36] (500 mg, 1.35 mmol) in
anhydrous ethanol (10 mL) were successively added propargyl
0.34 mmol) as a yellow solid. Yield 78%; mp 184e186 ꢁC; IR (KBr)
1178, 1351, 1466, 1516, 1676, 2200e2800, 2946, 3049, 3200e
3400 cmꢀ1; 1H NMR (200 MHz, DMSO-d6)
n
d
1.27 (t, 3H, J ¼ 7.1 Hz,
3
bromide (122
mL,1.62 mmol) and triethylamine (187
mL,1.35 mmol).
CH3), 3.33 (m, 4H, (CO)NHCH2CH2N, CH2CH3), 3.61 (qd, 2H, JHe
The mixture was stirred at room temperature for 72 h before
addition of water (33 mL). The resulting solution was then extrac-
ted with dichloromethane (3 ꢃ 33 mL). The organic layers were
combined, dried on magnesium sulphate, filtered and evaporated
under vacuum. The obtained residue was purified by chromatog-
raphy (Al2O3, CH2Cl2/EtOH, 99/1, v/v) to give compound 35
(522 mg, 1.28 mmol) as a beige solid. Yield 95%; Rf (Al2O3, CH2Cl2/
¼ 28.6 Hz, J ¼ 4.2 Hz, CH2CH2F), 3.78 (q, 2H, J ¼ 6.0 Hz, (CO)
F
2
NHCH2CH2N), 4.69 (br s, 5H), 4.92 (td, 2H, JHeF ¼ 47.5 Hz,
J ¼ 4.0 Hz, CH2F), 7.93 (d, 1H, J ¼ 8.8 Hz, H-8), 8.26 (dd, 1H, J ¼ 1.9,
8.8 Hz, H-7), 8.64 (d, 1H, J ¼ 1.9 Hz, H-5), 9.40 (t, 1H, J ¼ 6.0 Hz, NH),
9.46 (s, 1H, H-3), 10.89 (br s, 1H, NH); 13C NMR (50 MHz, DMSO-d6)
d
8.3, 33.9, 48.1, 50.7, 51.8 (d, 2JCeF ¼ 20 Hz), 78.7 (d, 1JCeF ¼ 164 Hz),
99.6, 130.7, 137.5, 138.9, 139.9, 143.6, 144.3, 144.4, 163.6; 19F NMR
EtOH, 99/1, v/v) 0.48; mp 135e137 ꢁC; IR (KBr)
1474, 1534, 1670, 2967, 3000e3600 cmꢀ1 1H NMR (200 MHz,
CDCl3)
n
1047, 1176, 1355,
(DMSO-d6)
d
ꢀ220.98 (tt, 2JHeF ¼ 47.3 Hz, 3JHeF ¼ 28.6 Hz); ESI-MS
;
m/z 417.0 [M þ H]þ. Anal. (C15H18OIN4F, 2HCl) calculated: C: 36.83;
d
1.12 (t, 3H, J ¼ 7.1 Hz, CH3), 2.23 (t, 1H, J ¼ 2.3 Hz, C^CH),
H: 4.12; N: 11.45; found: C: 36.59; H: 4.29; N: 11.07.
2.67 (q, 2H, J ¼ 7.1 Hz, CH2CH3), 2.84 (t, 2H, J ¼ 6.0 Hz, (CO)
NHCH2CH2N), 3.52 (d, 2H, J ¼ 2.3 Hz, CH2C^CH), 3.62 (q, 2H,
J ¼ 6.0 Hz, (CO)NHCH2CH2N), 7.78 (d, 1H, J ¼ 8.8 Hz, H-8), 8.02 (dd,
1H, J ¼ 1.9, 8.8 Hz, H-7), 8.32 (m, 1H, NH), 8.54 (d, 1H, J ¼ 1.9 Hz, H-
4.2.30. N-[2-[N-Ethyl-N-((E)-4-fluorobut-2-enyl)]amino]ethyl]-6-
iodoquinoxaline-2-carboxamide dihydrochloride salt (39)
This compound was synthesized according to the procedure
described for 38, starting from compound 17 (200 mg, 0.45 mmol).
Reaction time: 12 h at room temperature to give compound 39
(206 mg, 0.40 mmol) as a very hygroscopic beige solid. Yield 89%;
5), 9.59 (s, 1H, H-3); 13C NMR (50 MHz, acetone-d6)
d 13.2, 37.8, 41.9,
48.1, 52.8, 74.5, 79.3, 98.3, 131.9, 139.2, 140.3, 140.6, 145.2, 145.4,
145.6, 163.5; ESI-MS m/z 409.1 [M þ H]þ.
mp 139e141 ꢁC (dec.); IR (KBr)
n
1166, 1522, 1676, 2300e2600,
1.27 (t, 3H,
4.2.28. N-[2-[(N-Ethyl)-[[1-(2-fluoroethyl)-1H-(1,2,3)triazol-4-yl]
methyl]amino]ethyl]-6-iodoquinoxaline-2-carboxamide (37)
3200e3500 cmꢀ1 1H NMR (200 MHz, DMSO-d6)
;
d
J ¼ 7.1 Hz, CH3), 3.24 (m, 4H, (CO)NHCH2CH2N, CH2CH3), 3.75 (q, 2H,
J ¼ 5.9 Hz, (CO)NHCH2CH2N), 3.88 (m, 2H, NCH2CH¼CHCH2F), 4.94
(m, 1H, NH), 4.95 (dd, 2H, 2JHeF ¼ 46.6 Hz, J ¼ 4.6 Hz, CH2F), 5.96 (m,
1H, NCH2CH¼CHCH2F), 6.23 (tt, 1H, 4JHeF ¼ 15.8 Hz, J ¼ 4.6, 15.8 Hz,
NCH2CH¼CHCH2F), 7.93 (d, 1H, J ¼ 8.8 Hz, H-8), 8.26 (dd, 1H, J ¼ 1.5,
8.8 Hz, H-7), 8.64 (d, 1H, J ¼ 1.5 Hz, H-5), 9.39 (t, 1H, J ¼ 5.6 Hz, NH),
9.46 (s, 1H, H-3), 10.66 (br s, 1H, NH); 13C NMR (50 MHz, DMSO-d6)
Cesium fluoride (336 mg, 2.21 mmol) was dried by azeotropic
distillation with anhydrous acetonitrile (4 ꢃ 7 mL) at 90 ꢁC under
vacuum. After cooling to room temperature, a solution 2-azidoethyl
of p-toluenesulfonate (176 mg, 0.73 mmol), synthesized according
to the procedure described by Demko et al. [41], in a mixture of
anhydrous tert-amyl alcohol and acetonitrile (7.4 mL, 1/1, v/v) was
added under argon. The mixture was stirred at 100 ꢁC for 2 h to yield
2-fluoro-1-azidobutane (36). Back to room temperature, a 1.0 M
d
8.3, 33.9, 47.0, 49.7, 52.5, 81.9 (d, 1JCeF ¼ 162 Hz), 99.5,121.4 (d, 2JCe
¼ 14 Hz), 130.7, 135.0 (d, 3JCeF ¼ 17 Hz), 137.5, 138.9, 139.9, 143.5,
F
2
aqueous pentahydrate copper (II) sulphate solution (370
mL), a 1.0 M
144.2, 144.4, 163.5; 19F NMR (DMSO-d6)
d
ꢀ218.25 (tdd, JHe
3
4
aqueous ascorbic acid solution (370 L) and compound 35 (300 mg,
m
¼ 46.4 Hz, JHeF ¼ 16.5 Hz, JHeF ¼ 2.5 Hz); ESI-MS m/z 442.9
F
0.73 mmol) were successively added. The mixture was stirred at
room temperature for 12 h before addition of water (5 mL) and a
2.0 M aqueous sodium carbonate solution (5 mL). The solution was
extracted with dichloromethane (4 ꢃ 10 mL) and the organic layers
were combined, dried on magnesium sulphate, filtered and evapo-
rated under vacuum. The obtained residue was purified by chro-
matography (Al2O3, CH2Cl2/EtOH, 99/1, v/v) to give compound 37
(311 mg, 0.63 mmol) as a brown oil. Yield 86%; Rf (Al2O3, CH2Cl2/
[M þ H]þ. Anal. (C17H20OIN4F, 2HCl) calculated: C: 39.63; H: 4.30;
N: 10.88; found: C: 41.33; H: 4.56; N: 11.30.
4.2.31. N-[2-[[N-Ethyl-N-(4-fluorobut-2-ynyl)]amino]ethyl]-6-
iodoquinoxaline-2-carboxamide dihydrochloride salt (40)
This compound was synthesized according to the procedure
described for 38, starting from compound 20 (100 mg, 0.23 mmol).
Reaction time: 12 h at room temperature to give compound 40
(95 mg, 0.19 mmol) as a very hygroscopic yellow solid. Yield 81%;
EtOH, 99/1, v/v) 0.41; IR (CCl4)
n 1145, 1354, 1388, 1437, 1473, 1522,
1659,1673, 2837, 2971, 3246, 3386 cmꢀ1; 1H NMR (200 MHz, CDCl3)
mp 105e107 ꢁC; IR (KBr)
n
1165, 1356, 1474, 1534, 1672, 2400e2800,
1.29 (t, 3H,
d
1.12 (t, 3H, J ¼ 7.1 Hz, CH3), 2.64 (q, 2H, J ¼ 7.1 Hz, CH2CH3), 2.76 (t,
2900e3650 cmꢀ1 1H NMR (200 MHz, DMSO-d6)
;
d
2H, J ¼ 6.1 Hz, (CO)NHCH2CH2N), 3.59 (q, 2H, J ¼ 6.1 Hz, (CO)
J ¼ 7.0 Hz, CH3), 3.34 (m, 4H, (CO)NHCH2CH2N, CH2CH3), 3.80 (m,
3
2
NHCH2CH2N), 3.88 (s, 2H, NCH2C), 4.57 (td, 2H, JHeF ¼ 27.5 Hz,
2H, (CO)NHCH2CH2N), 4.38 (m, 2H, NCH2C^C), 5.17 (d, 2H, JHe
J ¼ 4.6 Hz, CH2CH2F), 4.71 (td, 2H, 2JHeF ¼ 51.0 Hz, J ¼ 4.6 Hz, CH2F),
7.65 (s,1H, C]CHCH2CH2F), 7.83 (d,1H, J ¼ 8.8 Hz, H-8), 8.05 (dd,1H,
J ¼ 1.8, 8.8 Hz, H-7), 8.40 (m,1H, NH), 8.57 (d,1H, J ¼ 1.8 Hz, H-5), 9.60
¼ 46.9 Hz, CH2F), 7.63 (br s, 2H, NH), 7.91 (d, 1H, J ¼ 8.8 Hz, H-8),
F
8.23 (dd, 1H, J ¼ 1.5, 8.8 Hz, H-7), 8.60 (d, 1H, J ¼ 1.5 Hz, H-5), 9.44
(m, 2H, H-3, NH), 11.58 (br s, 1H, NH); 13C NMR (50 MHz, DMSO-d6)
(s, 1H, H-3); 13C NMR (50 MHz, CDCl3)
d
12.1, 37.2, 47.6, 48.1, 50.4 (d,
d
8.7, 34.1, 41.1, 48.0, 50.6, 70.7 (d, JCeF ¼ 162 Hz), 79.0 (d, JCe
1
3
2JCeF ¼ 20 Hz), 51.5, 81.5 (d, JCeF ¼ 172 Hz), 98.0, 123.5 (d, JCe
¼ 12 Hz), 84.1 (d, JCeF ¼ 22 Hz), 99.6, 130.7, 137.5, 138.9, 139.8,
1
3
2
F

852
A. Maisonial et al. / European Journal of Medicinal Chemistry 63 (2013) 840e853
143.5,144.4,144.5,163.6; 19F NMR (DMSO-d6)
d
ꢀ218.61; ESI-MS m/
4.3.3. General method for purification
z 441.1 [M þ H]þ. Anal. (C17H18OIN4F, 2HCl) calculated: C: 39.79; H:
The vial cap and septumwere removed. The resulting suspension
was deposited on an extrelutÒ column. After 10 min, the columnwas
eluted with dichloromethane (5 ꢃ 3 mL). The collected organic ex-
tracts were evaporated under reduced pressure, taken up with
3.93; N: 10.92; found: C: 39.94; H: 4.04; N: 10.93.
4.2.32. N-(12-Ethyl-1-fluoro-3,6,9-trioxa-12-azatetradecan-14-yl)-
6-iodoquinoxaline-2-carboxamide dihydrochloride salt (41)
This experiment was carried out in a glove compartment, under
methanol (2 ꢃ 100
mL), and purified by RP-HPLC. Fractions con-
taining the expected radiolabelled product were collected, evapo-
rated to dryness, redissolved in dichloromethane (2 mL) and treated
with a 2.0 N hydrochloric acid solution in anhydrous ether (5 mL).
The resulting hydrochloride solution was evaporated under reduce
pressure, and the dry residue was suspended in anhydrous ether
(5 mL). The solvent was then evaporated under vacuum for 30 min to
give the radiolabelled tracer as corresponding hydrochloride salt.
The labelling conditions and characterization data for radio-
tracers [125I]14, [125I]34, and [125I]38e42 are given in Table 2.
For subsequent injection in mice, radiotracers [125I]38, [125I]39,
argon atmosphere. To a solution of compound 33 (10 mg,18.2 mmol)
in anhydrous dichloromethane (1 mL) was added a 2.0 N hydro-
chloric acid solution in anhydrous diethylether (2 mL) under argon.
After 5 min, the solvent was evaporated under a flux of argon. The
residue was suspended in anhydrous diethylether (2 mL) and dried
under a flux of argon to give dihydrochloride salt 41 (9.4 mg,
15.1
diamond accessory)
2953, 3300e3400 cmꢀ1
m
mol) as a very hygroscopic yellow solid. Yield 83%; IR (ATR
409, 1048, 1130, 1355, 1475, 1538, 1592, 1674,
1H NMR (200 MHz, CDCl3)
1.07 (t, 3H,
n
;
d
J ¼ 7.1 Hz, CH3), 3.37 (m, 6H, CH2CH3, NCH2CH2O, CONHCH2CH2N),
3.65 (m, 10H, 5CH2O), 3.77 (m, 2H, CH2CH2F), 4.02 (q, 2H, J ¼ 6.2 Hz,
CONHCH2CH2N), 4.54 (td, 2H, 2JHeF ¼ 47.7 Hz, J ¼ 4.2 Hz, CH2F), 7.96
(d, 1H, J ¼ 8.9 Hz, H-8), 8.08 (dd, 1H, J ¼ 1.9, 8.9 Hz, H-7), 8.59 (d, 1H,
J ¼ 1.6 Hz, H-5), 9.28 (br s, 1H, NH), 9.56 (s, 1H, H-3); 19F NMR
[
125I]40, [125I]41 and [125I]42 were prepared in solution in physio-
logical serum while [125I]14 and [125I]34, which were not converted
into corresponding hydrochloride salts, were dissolved in a solution
of ethanol 5% in physiological serum.
(CDCl3)
d
ꢀ222.76; ESI-MS m/z 549.1 [M
þ
H]þ; Anal.
4.4. In vivo scintigraphic imaging
(C21H30FIN4O4, 2HCl, 5H2O) calculated: C: 35.46; H: 5.95; N: 7.88;
found: C: 35.81; H: 6.23; N: 8.04.
In vivo scintigraphic imaging protocol was submitted and
approved by the french regional ethical animal use committee
(CEMEA, Auvergne, authorization no. CE18-09, 2010). Scintigraphic
imaging of radioiodinated compounds in mice was performed us-
4.2.33. N-[2-[(N-Ethyl)-[[1-(2-fluoroethyl)-1H-(1,2,3)triazol-4-yl]
methyl]amino]ethyl]-6-iodoquinoxaline-2-carboxamide
trihydrochloride salt (42)
This compound was synthesized according to the procedure
described for 38, starting from 37 (200 mg, 0.40 mmol). Reaction
time: 12 h at room temperature to give compound 42 (209 mg,
0.34 mmol) as a very hygroscopic yellow solid. Yield 86%; mp 98e
ing
a gamma camera dedicated to small animal imaging
(g
IMAGERÒ, Biospace Mesures, Paris, France). This camera consists
of a R 3292 Hamamatsu position-sensitive photomultiplier having a
continuous 4 mm thick ꢃ 120 mm diameter CsI (Na) crystal leading
to a 10 cm field of view. For [125I] imaging, the camera was equipped
with parallel-hole collimator 1.8/0.2/20 (hole diameter/septum
thickness/height in mm). All the acquisitions were performed with
a 15% window centred on the 35 keV peak of iodine-125.
100 ꢁC; IR (KBr)
n
1040, 1169, 1356, 1474, 1534, 1670, 2600e
1.34 (t, 3H, J ¼ 7.0 Hz,
3600 cmꢀ1; 1H NMR (200 MHz, DMSO-d6)
d
CH3), 3.15 (m, 2H, CH2CH3), 3.30 (m, 2H, (CO)NHCH2CH2N), 3.84 (m,
2H, (CO)NHCH2CH2N), 4.55 (m, 2H, CH2F), 4.70 (s, 2H, NCH2C), 4.89
(td, 2H, 3JHeF ¼ 20.9 Hz, J ¼ 4.4 Hz, CH2F), 5.36 (m, 2H, NH), 7.92 (d,
1H, J ¼ 8.8 Hz, H-8), 8.24 (dd,1H, J ¼ 1.6, 8.8 Hz, H-7), 8.47 (s,1H, C]
CHCH2CH2F), 8.62 (d, 1H, J ¼ 1.6 Hz, H-5), 9.44 (m, 2H, H-3, NH),
On day 14 after tumour implant, each [125I]-labelled compound
was administered intravenously viathetail veinwith 3.7 MBq/mouse
(range 3e8 animals/compound). At different time points after
radiotracer administration (1 h, 3 h, 6 h, 24 h, 72 h, 5 d, 7 d, 10 d and
14 d), mice were anaesthetized by intraperitoneal (i.p.) administra-
11.08 (br s, 1H, NH); 13C NMR (50 MHz, DMSO-d6)
d 8.6, 34.1, 45.4,
47.3, 50.2 (d, 2JCeF ¼ 19 Hz), 50.3, 81.8 (d, 1JCeF ¼ 167 Hz), 99.6, 127.6
tion (200 mL/mouse) of a ketamineexylazine mixture in saline: ke-
(d, 3JCeF ¼ 12 Hz), 130.7, 136.1, 137.5, 138.9, 139.8, 143.6, 144.3, 144.4,
tamine (100 mg/kg, Imalgene, Rhône Mérieux, Lyon, France) and
xylazine (10 mg/kg, Rompun, Bayer, France). A 10 min duration image
was acquired on anesthetized mice placed in prone position over the
collimator of the camera. Reproducibility in animal positioning for
the serial images was achieved using a graduated reference grid.
The injected activity to each mouse was also determined from
scintigraphic imaging of the syringe before and after the injection of
the radiotracer. Quantitative analysis of scintigraphic scans was
performed using the GAMMAVISIONþÒ software (Biospace Mesures,
Paris, France). For each scintigram, regions of interest (ROIs) were
manually drawn around the tumour, the whole body area, and the
controlateral muscle which was chosen as background. For each ROI,
total activity, min and max pixel values, ROI size (in mm2), average
activity per mm2 and standard deviation were obtained. Activities
were normalized to the injected activity and corrected for the
radioactive decay. For the tumour, the activity was also normalized to
the tumour weight as previously described [43] and expressed as
percentage of injected dose per gram of tumour (%ID/g).
163.6; 19F NMR (DMSO-d6)
d
ꢀ223.95; ESI-MS m/z 498.1 [M þ H]þ.
Anal. (C18H21OIN7F, 3HCl, H2O) calculated: C: 34.61; H: 4.20; N:
15.70; found: C: 37.74; H: 4.61; N: 15.49.
4.3. Radiochemistry
4.3.1. Method A
To a solution of the appropriate compound (2e4 mg) in acetic
acid (600
(25 G, 0.5 ꢃ 16 mm), a copper sulphate solution in acetic acid
(100
L, 1 mg mLꢀ1) used as catalyst and [125I]NaI (119e333 MBq).
The reaction mixture was heated at 130 ꢁC for 25e45 min. Back to
room temperature, the residue was taken up in water (500 L) and
L) was added.
mL) were added, in a sealed vial equipped with a needle
m
m
a 1.0 N aqueous sodium hydroxide solution (100
m
4.3.2. Method B
To a solution of the appropriate compound (2e4 mg) in citrate
buffer pH ¼ 4 (500 L) were added, in a sealed vial equipped with a
needle (25 G, 0.5 ꢃ 16 mm), an aqueous copper sulphate solution
(0.5 mg, 100
L) and [125I]NaI (181e363 MBq). The reaction mixture
was heated at 130 ꢁC for 25e30 min. After cooling to room tem-
perature, the residue was taken up in water (500 L) and a 1.0 N
aqueous sodium hydroxide solution (100 L) was added.
m
Acknowledgements
m
We would like to thank the CYCLOPHARMA SA Laboratories, the
CLARA (Cancéropôle Lyon Rhône-Alpes Auvergne), the ANR (French
Research Agency), the EMIL (European Molecular Imaging
m
m

A. Maisonial et al. / European Journal of Medicinal Chemistry 63 (2013) 840e853
853
[21] D. Denoyer, I. Greguric, P. Roselt, O.C. Neels, N. Aide, S.R. Taylor, A. Katsifis,
D.S. Dorow, R.J. Hicks, J. Nucl. Med. 51 (2010) 441e447.
[22] D. Denoyer, T. Potdevin, P. Roselt, O.C. Neels, L. Kirby, I. Greguric, A. Katsifis,
D.S. Dorow, R.J. Hicks, J. Nucl. Med. 52 (2011) 115e122.
[23] I. Greguric, S.R. Taylor, D. Denoyer, P. Ballantyne, P. Berghofer, P. Roselt,
T.Q. Pham, F. Mattner, T. Bourdier, O.C. Neels, D.S. Dorow, C. Loc’h, R.J. Hicks,
A. Katsifis, J. Med. Chem. 52 (2009) 5299e5302.
[24] S. Garg, K. Kothari, S.R. Thopate, A.K. Doke, P.K. Garg, Bioconjug. Chem. 20
(2009) 583e590.
Laboratories), the Bullukian Foundation, the French League against
Cancer, the French Directorate-General for Research & Innovation, the
AuvergneRegionCouncil, theGeneralCouncilofPuydeDôme, andthe
European Regional Development Fund for their financial support.
Appendix A. Supplementary data
[25] G. Ren, Z. Miao, H. Liu, L. Jiang, N. Limpa-Amara, A. Mahmood, S.S. Gambhir,
Z. Cheng, J. Nucl. Med. 50 (2009) 1692e1699.
[26] L. Rbah-Vidal, A. Vidal, S. Besse, F. Cachin, M. Bonnet, L. Audin, S. Askienazy,
F. Dollé, F. Degoul, E. Miot-Noirault, N. Moins, P. Auzeloux, J.M. Chezal, Eur.
J. Nucl. Med. Mol. Imaging (2012), http://dx.doi.org/10.1007/s00259-012-
2168-y.
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2012.11.
047. These data include MOL files and InChiKeys of the most
important compounds described in this article.
[27] M. Bonnet-Duquennoy, J. Papon, F. Mishellany, P. Labarre, J.L. Guerquin-Kern,
T.D. Wu, M. Gardette, J. Maublant, F. Penault-Llorca, E. Miot-Noirault, A. Cayre,
J.C. Madelmont, J.M. Chezal, N. Moins, Int. J. Cancer 125 (2009) 708e716.
[28] J.M. Chezal, J. Papon, P. Labarre, C. Lartigue, M.J. Galmier, C. Decombat,
O. Chavignon, J. Maublant, J.C. Teulade, J.C. Madelmont, N. Moins, J. Med.
Chem. 51 (2008) 3133e3144.
[29] M. Bonnet, F. Mishellany, J. Papon, A. Cayre, F. Penault-Llorca, J.C. Madelmont,
E. Miot-Noirault, J.M. Chezal, N. Moins, Pigm. Cell Melanoma Res. 23 (2010)
e1e11.
[30] J.L. Joyal, J.A. Barrett, J.C. Marquis, J. Chen, S.M. Hillier, K.P. Maresca, M. Boyd,
K. Gage, S. Nimmagadda, J.F. Kronauge, M. Friebe, L. Dinkelborg, J.B. Stubbs,
M.G. Stabin, R. Mairs, M.G. Pomper, J.W. Babich, Cancer Res. 70 (2010) 4045e
4053.
[31] J.M. Chezal, F. Dollé, J.C. Madelmont, A. Maisonial, E. Miot-Noirault, N. Moins, J.
Papon, B. Kuhnast, B. Tavitian, R. Boisgard, World Patent WO 2009095872,
2009.
[32] A. Maisonial, B. Kuhnast, J. Papon, R. Boisgard, M. Bayle, A. Vidal, P. Auzeloux,
L. Rbah, M. Bonnet-Duquennoy, E. Miot-Noirault, M.J. Galmier, M. Borel,
S. Askienazy, F. Dolle, B. Tavitian, J.C. Madelmont, N. Moins, J.M. Chezal, J. Med.
Chem. 54 (2011) 2745e2766.
[33] B. Cohen, E.R.V. Artsdalen, J. Harris, J. Am. Chem. Soc. 70 (1948) 281e285.
[34] O.C. Dermer, G.E. Ham, Ethylenimine and Other Aziridines, Chemistry and
Applications, Academic Press, New York, 1969.
[35] M. Glasser, E.G. Robins, J. Labelled Compd. Radiopharm. 52 (2009) 407e414.
[36] D. Denoyer, P. Labarre, J. Papon, E. Miot-Noirault, M.J. Galmier, J.C. Madelmont,
J.M. Chezal, N. Moins, J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 875
(2008) 411e418.
References
[1] V. Gray-Schopfer, C. Wellbrock, R. Marais, Nature 445 (2007) 851e857.
[2] S.N. Markovic, L.A. Erickson, R.D. Rao, R.H. Weenig, B.A. Pockaj, A. Bardia,
C.M. Vachon, S.E. Schild, R.R. McWilliams, J.L. Hand, S.D. Laman,
L.A. Kottschade, W.J. Maples, M.R. Pittelkow, J.S. Pulido, J.D. Cameron,
E.T. Creagan, Mayo Clin. Proc. 82 (2007) 490e513.
[3] S.N. Markovic, L.A. Erickson, R.D. Rao, R.H. Weenig, B.A. Pockaj, A. Bardia,
C.M. Vachon, S.E. Schild, R.R. McWilliams, J.L. Hand, S.D. Laman,
L.A. Kottschade, W.J. Maples, M.R. Pittelkow, J.S. Pulido, J.D. Cameron,
E.T. Creagan, Mayo Clin. Proc. 82 (2007) 364e380.
[4] R.M. MacKie, A. Hauschild, A.M. Eggermont, Ann. Oncol. 20 (Suppl. 6) (2009)
vi1e7.
[5] D.S. Rigel, J. Russak, R. Friedman, CA Cancer J. Clin. 60 (2010) 301e316.
[6] J.E. Gershenwald, M.I. Ross, N. Engl. J. Med. 364 (2011) 1738e1745.
[7] M.I. Ross, Semin. Cutan. Med. Surg. 29 (2010) 238e248.
[8] S.Q. Wang, P. Hashemi, Semin. Cutan. Med. Surg. 29 (2010) 174e184.
[9] J. Lutzky, Semin. Cutan. Med. Surg. 29 (2010) 249e257.
[10] C. Garbe, T.K. Eigentler, U. Keilholz, A. Hauschild, J.M. Kirkwood, Oncologist 16
(2011) 5e24.
[11] S. Dalle, N. Poulalhon, L. Thomas, N. Engl. J. Med. 365 (2011) 1448e1449.
[12] J.A. Sosman, K.B. Kim, L. Schuchter, R. Gonzalez, A.C. Pavlick, J.S. Weber,
G.A. McArthur, T.E. Hutson, S.J. Moschos, K.T. Flaherty, P. Hersey, R. Kefford,
D. Lawrence, I. Puzanov, K.D. Lewis, R.K. Amaravadi, B. Chmielowski,
H.J. Lawrence, Y. Shyr, F. Ye, J. Li, K.B. Nolop, R.J. Lee, A.K. Joe, A. Ribas, N. Engl.
J. Med. 366 (2012) 707e714.
[37] J.H. Jones, W.J. Holtz, E.J. Cragoe, J. Med. Chem. 16 (1973) 537e542.
[38] F. Dolle, P. Emond, S. Mavel, S. Demphel, F. Hinnen, Z. Mincheva, W. Saba,
H. Valette, S. Chalon, C. Halldin, J. Helfenbein, J. Legaillard, J.C. Madelmont,
J.B. Deloye, M. Bottlaender, D. Guilloteau, Bioorg. Med. Chem. 14 (2006)
1115e1125.
[39] H.J. Shine, P. Rangappa, J.N. Marx, D.C. Shelly, T. Ould-Ely, K.H. Whitmire,
J. Org. Chem. 70 (2005) 3877e3883.
[40] T. Kan, Y. Kita, Y. Morohashi, Y. Tominari, S. Hosoda, T. Tomita, H. Natsugari,
T. Iwatsubo, T. Fukuyama, Org. Lett. 9 (2007) 2055e2058.
[41] Z.P. Demko, K.B. Sharpless, Org. Lett. 3 (2001) 4091e4094.
[42] R. Bejot, T. Fowler, L. Carroll, S. Boldon, J.E. Moore, J. Declerck, V. Gouverneur,
Angew. Chem. Int. Ed. Engl. 48 (2009) 586e589.
[43] E. Miot-Noirault, J. Papon, M. Gardette, M. Bonnet-Duquennoy, P. Labarre,
A. Maisonial, J.C. Madelmont, J. Maublant, J.M. Chezal, N. Moins, Cancer Bio-
ther. Radiopharm. 24 (2009) 629e636.
[44] W.L.F. Armarego, D.D. Perrin, Purification of Laboratory Chemicals, fourth ed.,
Elsevier, 1997.
[45] G. Eglington, M.C. Whiting, J. Chem. Soc. (1950) 3650e3655.
[13] R.N. Amaria, K.D. Lewis, A. Jimeno, Drugs Today (Barc) 48 (2012) 109e118.
[14] R.M. Ings, Drug Metabol. Rev. 15 (1984) 1183e1212.
[15] D. Oltmanns, M. Eisenhut, W. Mier, U. Haberkorn, Curr. Med. Chem. 16 (2009)
2086e2094.
[16] M. Eisenhut, W.E. Hull, A. Mohammed, W. Mier, D. Lay, W. Just, K. Gorgas,
W.D. Lehmann, U. Haberkorn, J. Med. Chem. 43 (2000) 3913e3922.
[17] X. Liu, T.Q. Pham, P. Berghofer, J. Chapman, I. Greguric, P. Mitchell, F. Mattner,
C. Loc’h, A. Katsifis, Nucl. Med. Biol. 35 (2008) 769e781.
[18] J.M. Michelot, M.F. Moreau, A.J. Veyre, J.F. Bonafous, F.J. Bacin, J.C. Madelmont,
F. Bussiere, P.A. Souteyrand, L.P. Mauclaire, F.M. Chossat, et al., J. Nucl. Med. 34
(1993) 1260e1266.
[19] N. Moins, M. D’Incan, J. Bonafous, F. Bacin, P. Labarre, M.F. Moreau, D. Mestas,
E. Noirault, F. Chossat, E. Berthommier, J. Papon, M. Bayle, P. Souteyrand,
J.C. Madelmont, A. Veyre, Eur. J. Nucl. Med. Mol. Imaging 29 (2002) 1478e
1484.
[20] T.Q. Pham, I. Greguric, X. Liu, P. Berghofer, P. Ballantyne, J. Chapman,
F. Mattner, B. Dikic, T. Jackson, C. Loc’h, A. Katsifis, J. Med. Chem. 50 (2007)
3561e3572.