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A. Monaco et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6068–6072
Scheme 2. Synthesis of the trimethylammonium trifluoromethanesulfonate precursors (8) (A) and (11) (B). Reagents and conditions: (a) 2, EDC, HOBt, DCM, rt, overnight,
90%; (b) methyl trifluoromethanesulfonate, DCM, 2 h, 0 °C, 66%; (c) (i) NaH, THF, 5 min, rt; (ii) MeI, THF, 24 h, 73%; (d) methyl trifluoromethanesulfonate, DCM, 0 °C, 68%.
Scheme 3. Synthesis [18F]FPPA ([18F]-3). Reagents and conditions: (a) [18F]FÀ, K222/K2CO3, CH3CN or DMSO, 60–165 °C, 10–15 min, 0.4–5%; (b) 4, water/THF, CuSO4 (0.3 M),
sodium ascorbate (0.7 M), rt, 15 min, 66%; (c) [18F]FÀ, K222/K2CO3, CH3CN, 10 min, 95 °C, 69%; (d) (i) NaBH4, MeOH, Pd/C, rt, 2 min; (ii) 12 M HCl, rt, 53%; (e) 13, Et3N, DCM,
75 °C, rt, 10 min, 44%.
Treatment of
6
with 4-pentynoic acid gave N-(4-(dimethyl-
of [18F]fluoro-nitrobenzene (12) with sodium borohydride, in pres-
ence of palladium on activated carbon, gave 4-[18F]fluoroaniline
([18F]-1) as an hydrochloric salt after quenching the reaction with
either 1 M HCl or 12 M HCl, with a radiochemical yields of 45% and
53% (decay corrected), respectively.25 Identity of [18F]-1 was con-
firmed by HPLC analysis by co-elution with authentic unlabeled
4-fluoroaniline (Fig. 1-SD). Subsequent coupling of 4-[18F]fluoroan-
iline with the 2,5-dioxopyrrolidin-1-yl pent-4-ynoate (13)26 in
dichloromethane in presence of triethylamine afforded the 18F-la-
beled prosthetic group [18F]-3 in 44% radiochemical yield (decay
corrected) within 40 min (Fig. 2-SD).
amino)phenyl)pent-4-ynamide (7) in 90% yield, while dimethyla-
tion of 9 with iodomethane yielded N,N-dimethyl-4-nitroaniline
(10). Finally, methylation of the dimethylamino intermediates 7
and 10 with methyl trifluoromethanesulfonate afforded the trime-
thylammonium precursors 8 and 11 in 66% and 68% yield,
respectively.
The radiolabeled RGD peptide [18F]-5 or [18F]FPPA-c(RGDfK)
was prepared starting with the standard kryptofix–K2CO3-medi-
ated nucleophilic 18F-exchange reaction with the trimethylammo-
nium triflate precursors 8 or 11 (Scheme 3). Introduction of the
fluorine-18 using a no-carrier-added nucleophilic substitution
with K[18F]FÀ/K222 was initially conducted with precursor 8 in ace-
tonitrile at 60 °C for 10 min, but low radiochemical yield (0.4% de-
cay noncorrected) was observed. Therefore, we investigated
whether we could improve the nucleophilic incorporation of
Finally, [18F]-3 was conjugated with c(RGDfK(N3)) following the
same procedure used for the preparation of the nonradioactive
conjugate (5), to obtain the 18F-labeled peptide [18F]-5 with 66%
yield (decay corrected) (Scheme 3). [18F]FPPA-c(RGDfK) was then
obtained in about 140 min with a total radiochemical yield of
29% (decay corrected). Identity of the new radiopharmaceutical
was confirmed by comparing its HPLC mobility with the retention
time of the nonradioactive analogue (Fig. 3-SD).
The affinity of the fluorinated c-(RGDfK) was then experimen-
tally tested with an ELISA in vitro assay using recombinant solu-
ble integrins binding to their immobilized natural ligands in the
absence or presence of gradual amounts of the test peptide. Sol-
[
18F]fluoride into this trimethylammonium triflate salt by changing
the reaction conditions (temperature, time, and solvent). We ob-
tained our best radiochemical yield of 4.9% (decay noncorrected)
with respect to initial
[
18F]fluoride when the reaction was
performed in DMSO at 165 °C for 12 min. The lack of electron with-
drawing groups on the aromatic ring that would favor the fluorina-
tion at the 4-position considerably limited the efficacy of this
reaction. Therefore, although this one step approach would obviate
the need of sophisticated radiochemical route to prepare [18F]FPPA,
we considered that it would not provide quantities which are prac-
tical for use as PET radiopharmaceuticals. Consequently, a three
steps radiochemical route has been established. The first radio-
chemical step (Scheme 3B), which consisted of the 18F-fluorination
of our second activated trimethylammonium precursor 11 resulted
in [18F]fluoro-nitrobenzene (12), with radiochemical yield of 69%
(decay noncorrected) when the reaction mixture was heated at
95 °C for 10 min in acetonitrile. Then, reduction of the nitro group
uble recombinant
HEK293T cells transfected with cDNA encoding for the
a
vb3 and
a
5b1 integrins were expressed in
and b
a
subunits of the integrins of interest.27 All cDNAs encoded for
truncated integrins (i.e., all integrin subunits lacked their respec-
tive transmembrane and C-terminal domains) and were tagged
with either a His-tag or fused with a human IgG-Fc-fragment.
As a result expressed integrins were secreted in the culture med-
ium (‘conditioned medium’). Soluble truncated
5b1 was used for specificity control) are assumed to adopt a
rod-like shape conformation associated with high affinity
avb3 and a5b1
(
a