Synthesis of a non-peptidic PET tracer designed for α 5β1 integrin receptor

Article abstract of DOI:10.1002/jlcr.3190

Arginine-glycine-aspartic acid (RGD)-containing peptides have been traditionally used as PET probes to noninvasively image angiogenesis, but recently, small selective molecules for α₅β₁ integrin receptor have been developed with prom

Full text of DOI:10.1002/jlcr.3190

Research Article
Received 2 December 2013,
Revised 16 January 2014,
Accepted 23 January 2014
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3190
Synthesis of a non-peptidic PET tracer
designed for α5β1 integrin receptor
Alessandra Monaco,a,b Olivier Michelin,c John Prior,d Curzio Rüegg,e
Leonardo Scapozza,b and Yann Seimbillea,b
*
Arginine–glycine–aspartic acid (RGD)-containing peptides have been traditionally used as PET probes to noninvasively image
angiogenesis, but recently, small selective molecules for α5β1 integrin receptor have been developed with promising results.
Sixty-one antagonists were screened, and tert-butyl (S)-3-(2-((3R,5S)-1-(3-(1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)propanoyl)-5-
((pyridin-2-ylamino)methyl)pyrrolidin-3-yloxy)acetamido)-2-(2,4,6-trimethylbenzamido)propanoate (FPMt) was selected for
the development of a PET tracer to image the expression of α5β1 integrin receptors. An alkynyl precursor (PMt) was initially
synthesized in six steps, and its radiolabeling was performed according to the azide–alkyne copper(II)-catalyzed Huisgen’s
cycloaddition by using 1-azido-2-[18F]fluoroethane ([18F]12). Different reaction conditions between PMt and [18F]12 were
investigated, but all of them afforded [18F]FPMt in 15min with similar radiochemical yields (80–83%, decay corrected). Overall,
the final radiopharmaceutical ([18F]FPMt) was obtained after a synthesis time of 60–70min in 42–44% decay-corrected
radiochemical yield.
Keywords: α5β1 integrin receptor; PET, 1-azide-2-[18F]fluoroethane; click chemistry; peptido-mimetic
reliable and reproducible procedure to radiolabel this pyrrolidine
Introduction
derivative that relies on the following: (i) the preparation and
purification of a widely used radiolabeled synthon 1-azido-2-[18F]
Integrins are an extensive group of transmembrane cell
adhesion receptors, composed by noncovalently linked α and β
fluoroethane and (ii) its subsequent conjugation to our alkynyl
precursor (PMt) via the Huisgen’s 1,3-dipolar cycloaddition.
subunits.1 They mediate cell migration and cell proliferation by
the connection of the extracellular matrix with the intracellular
cytoskeleton.2 Activation of the integrins by ligand binding
Experimental
promotes cell proliferation, migration, and survival, but unligated
or antagonized integrins may activate ‘integrin-mediated
Reagents and instrumentation
death’.3 Integrins are of crucial support for the formation of
capillaries and angiogenesis in physiological and pathological
processes,4 and three of them (αvβ3, αvβ5, and α5β1) have a
prominent role for the development of a new vascular system.5,6
The involvement of these integrins in many diseases such as
tumor, thrombosis, cardiovascular, and inflammatory diseases
makes them an appealing target for the development of
antiangiogenic therapies.7,8 Of them, αvβ3 is the most targeted
integrin, and several antagonists such as monoclonal
antibodies9,10 and arginine–glycine–aspartic acid (RGD)-based
peptides11 are currently in clinical trials.7,12,13 Moreover,
radiolabeled analogs of the RGD peptides have been evaluated
in preclinical and clinical studies, as tracers, to visualize
angiogenesis in growing tumors.14,15
All the solvents and reagents were purchased from Sigma-Aldrich and Fluka
(Basel, Switzerland) or VWR (Nyon, Switzerland) and were used without
further purification. Flash chromatography columns were performed on
VWR silica gel (0.063–0.200 mm). Thin layer chromatography (TLC) was
performed on precoated silica gel 60F254 plastic sheets from VWR. The
compounds were monitored under ultraviolet (UV) light at 254 nm or by
brief immersion in potassium permanganate solution. Radioactive spots
aCyclotron Unit, University Hospital of Geneva, Rue Gabrielle Perret-Gentil 4, 1211
Geneva, Switzerland
bSchool of Pharmaceutical Sciences, University of Geneva and University of
Lausanne, Quai Ernest Ansermet 30, 1211 Geneva, Switzerland
“”In the last years, integrin α5β1 has gained great interest, not
only because of its involvement in tumor angiogenesis but also
for its fundamental role in brain angiogenesis.16 Recently, several
small molecules have been developed to selectively antagonize
this integrin receptor.17–19 The promising biological results
obtained have enhanced the interest in non-αv integrins as target
for new therapies and for imaging to monitor tumor angiogenesis
and neurovascular remodeling after ischemic stroke.16,20
cSwiss Institute of Bioinformatics, University of Lausanne, Quartier Sorge, 1015
Lausanne, Switzerland
dCHUV, Department of Nuclear Medicine, Rue du Bugnon 46, 1011 Lausanne,
Switzerland
eFaculty of Science, Department of Medicine, University of Fribourg, Ch. du Musée
8, 1700 Fribourg, Switzerland
*Correspondence to: Yann Seimbille, Cyclotron Unit, University Hospital of
Geneva, Rue Gabrielle Perret-Gentil 4, 1211 Geneva, Switzerland.
E-mail: Yann.Seimbille@hcuge.ch
In this manuscript, we advance a novel nonpeptidic PET tracer
designed to image the α5β1 integrin receptor. We report herein a
J. Label Compd. Radiopharm 2014
Copyright © 2014 John Wiley & Sons, Ltd.

A. Monaco et al.
were detected with a Cyclone Phosphorimager from PerkinElmer (Waltham, concentrated, and 20 mL of water was added. The aqueous layer was
Massachusetts). The radiochemical yields were determined by TLC analysis extracted with ethyl acetate (3 × 30 mL). The organic layer was dried over
of reaction mixture samples taken at specified times. High-performance
Na2SO4, filtered, and concentrated under vacuum. The crude product
liquid chromatography (HPLC) was performed on an UltiMate 3000 Rapid was purified by chromatography on silica gel using EtOAc/hexane (1:1)
Separation LC system (Dionex, Basel, Switzerland) with Chromeleon 6.8 to obtain light yellow oil. Yield: 88%. 1H-NMR (300 MHz, CDCl3) δ: 4.96
software package. The UV detector was set at four different wavelengths (1H, d, J = 8.1 Hz), 4.30 (1H, q, J = 8.5 Hz), 4.20 (1H, t, J = 1,8 Hz), 3.95 (2H,
(215nm, 220nm, 254nm, and 280 nm). Radioactivity was detected with a d, J = 5.4 Hz), 3.76–3.5 (4H, m), 2.62–2.47 (4H, m), 2.26–2.18 (1H, m),
NaI scintillation detector (Nuclear Interface, Munich, Germany) connected 1.73–1.64 (2H, m), and 1.46 (9H, s). 13C-NMR (300 MHz, CDCl3) δ: 172.07,
on the outflow of the UV detector. The HPLC column used for the 169.25, 82.08, 77.42, 77.21, 69.00, 68.70, 66.68, 60.13, 53.61, 34.12, 33.86,
purification of the product and the characterization of radioactive 28.07 (3C), and 14.86. MS (ESI): m/z 312.1 [M + H]+.
compounds against standards was a Phenomenex Gemini 5-μ C18 110-Å
tert-Butyl 2-((3R,5S)-5-((tert-butyldimethylsilyl)oxy)methyl)-1-(pent-
4-ynoyl)pyrrolidin-3-yl)oxy)acetate (5)
(250× 4.60 mm) column operated at a flow rate of 1 mL/min using a
gradient of 5% acetonitrile in water containing 0.1% trifluoroacetic acid
(TFA) to CH3CN/TFA (99.9:0.1) over 20min. The radiochemical purities were
determined by HPLC analyses. NMR data were recorded with a Varian
Gemini 2000 NMR Spectrometer and Oxford 300 Nuclear Magnetic
Instrument (Oxfordshire, UK). The samples were dissolved either in CDCl3
or in acetone-d6 (Cambridge Isotope Laboratories Inc., Burgdorf,
Switzerland), and the data were processed by MestRe-C 4.8 software.
Chemical shifts (δ) are expressed in ppm relative to the signals of the
solvents and coupling constants (J) in Hz. Mass spectrometry (MS) analyses
were performed using an ESI-MS instrument, API 150EX from AB/MDS
(Sciex, Framingham, Massachusetts), or ES TQ-detector Aqui™ (Waters,
Baden-Dättwil, Switzerland). High-resolution mass spectrometry (HRMS)
spectra were recorded by ESI/nanoESI-IT Esquire 3000 plus (Bruker,
Fällanden, Switzerland).
4 (0.80 g, 2.57 mmol), imidazole (0.44 g, 6.42 mmol), and tert-butyldimethylsilyl
chloride (0.43 g, 2.83 mmol) were dissolved in 15 mL of dimethylformamide
(DMF) and stirred for 3 h at room temperature. The solvent was removed
by evaporation, and 20 mL of EtOAc was added and extracted with water,
saturated NaHCO3, and dried over Na2SO4, then filtered. The solvent was
removed by evaporation, and the product was purified by chromatography
on silica gel using EtOAc to obtain colorless oil. Yield: 82%. 1H-NMR
(300 MHz, CDCl3) δ: 4.37–4.24 (2H, m), 3.96–3.89 (3H, m), 3.68–3.63 (1H,
dd, Jd = 4.2, Jd = 6 Hz), 3.58–3.51 (2H, m), 2.59–2.41 (4H, m), 2.26–2.17
(1H, m), 2.07–1.95 (2H, m), 1.47 (9H, s), 0.85 (9H, s), and 0.04–0.01 (6H, m).
13C-NMR (300 MHz, CDCl3) δ: 169.40, 156.50, 81.88, 78.43, 78.35, 68.60,
67.08 (2C), 63.11, 57.73, 52.91, 33.91, 33.27, 28.21 (3C), 25.85 (3C), 14.06,
and À5.50 (2C). MS (ESI): m/z 426.5 [M+ H]+.
tert-Butyl (S)-3-(2-((3R,5S)-5-(hydroxymethyl)-1-(pent-4-ynoyl)pyrrolidin-3-
yloxy)acetamido)-2-(2,4,6-trimethylbenzamido)propanoate (6)
Chemistry
Methyl (2S,4R)-4-hydroxy-1-(pent-4-ynoyl)pyrrolidine-2-carboxylate (2)
5 (0.10 g, 2.35 mmol) was dissolved in 25 mL of MeOH, and LiOH * H2O
(0.23 g, 9.40 mmol) was added. After stirring for 4 h at room temperature,
NH4Cl was added, and the solution was stirred for 10 min. The solid was
filtered, the solvent was removed under vacuum, and then, 30 mL of
EtOAc were added and extracted with water (2 × 30 mL). The organic
layer was dried over Na2SO4 and filtered, and the solvent was removed
under reduced pressure. The crude was dissolved in 2 mL of DMF; 9
(0.92 g, 2.99 mmol), EDC (0.54 g, 2.82 mmol), and DMAP (0.43 g,
3.53 mmol) were added; and the solution was stirred overnight at room
temperature. The solvent was then removed under reduced pressure,
20 mL of ethyl acetate was added, and the organic layer was washed with
saturated NaHCO3 (2 × 20 mL) and brine (20 mL). The organic layer was
dried with Na2SO4 and filtered, and the solvent removed under reduced
pressure. The product was purified by column chromatography on silica
gel using EtOAc/hexane (2:3) to obtain light brown oil. Yield: 40%. 1H-
NMR (300 MHz, CDCl3) δ: 7.39 (1H, td, J = 31.2 Hz), 6.79 (3H, s), 4.78–4.71
(1H, m), 4.20–4.09 (2H, m), 3.95–3.81 (8H, m), 2.57–2.31 (4H, m), 2.23–
2.22 (9H, m), 2.14 (3H, s), 1.76–1.65 (1H, m), and 1.45 (9H, m). 13C-NMR
(300 MHz, CDCl3) δ: 171.68, 171.24, 169.56, 168.60, 138.63, 133.88 (2C),
133.66, 128.15 (2C), 83.27, 77.90, 77.68, 68.76, 68.52, 67.91, 65.55, 59.48,
52.97, 42.26, 33.83, 33.36, 27.81 (3C), 20.94, 18.88 (2C), and 13.86. MS
(ESI): m/z 544.7 [M + H]+.
Methyl-(2S,4R)-4-hydroxypyrrolidie-2-carboxylate (1) (1.00 g, 5.48 mmol),
4-pentynoic acid (0.54 g, 5.48 mmol), 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide (EDC) (1.26 g, 6.57 mmol), and 4-dimethylaminopyridine
(DMAP) (1.00 g, 8.21 mmol) were dissolved in 20 mL of dichloromethane
(DCM). Et3N (0.20 mL) was added dropwise, and the reaction mixture
was stirred at room temperature overnight. Then, the mixture was
extracted with EtOAc (3 × 30 mL). The organic layer was dried over
Na2SO4, filtered, and concentrated under reduced pressure. The crude
product was purified by chromatography on silica gel hexane/EtOAc
1
(1:1) to obtain colorless oil. Yield: 90%. H-NMR (300 MHz, CDCl3) δ: 4.42
(1H, d, J = 10.2 Hz), 4.39 (2H, t, J = 7.7 Hz), 4.21 (1H, bs), 3.66–3.59 (4H,
m), 2.48–2.35 (4H, m), 2.16 (1H, t, J = 10.65 Hz), and 1.95–1.86 (2H, m).
13C-NMR (300 MHz, CDCl3) δ: 172.75, 170.35, 83.44, 70.53, 69.03, 57.77,
55.16, 52.59, 38.02, 33.69, and 14.17. MS (ESI): m/z 226.4 [M + H]+.
Methyl (2S,4R)-4-(2-tert-butoxy)-2-oxoethoxy)-1-(pent-4-ynoyl)pyrroli
dine-2-carboxylate (3)
NaH 60% in paraffin oil (0.31 g, 7.75 mmol), tert-butylbromoacetate (2.00 mL,
13.67 mmol), and n-Bu4N+IÀ (1.44 g, 3.90 mmol) were suspended under
argon in 28mL of dry THF. Then, 2 (1.10 g, 4.88 mmol) was dissolved in
2 mL of dry THF, slowly added to the reaction mixture, and stirred overnight.
The solvent was removed under reduced pressure. The crude product was
purified by chromatography on silica gel using EtOAc/hexane (2:3) to obtain
light yellow oil. Yield: 75%. 1H-NMR (300 MHz, CDCl3) δ: 4.53 (1H, t,
J= 7.65 Hz), 4.28 (1H, q, J= 4.3 Hz), 3.96 (2H, d, J= 2.1Hz), 3.77 (1H, s), 3.72
(2H, s), 3.65 (1H, dd, Jd = 1, Jd =3Hz), 2.57–2.50 (4H, m), 2.41–2.22 (1H, m),
2.16–2.03 (1H, m), 1.96 (1H, t, J= 2.4Hz), 1.68 (1H, s), and 1.47 (9H, s). 13C-
NMR (300 MHz, CDCl3) δ: 172.64, 169.77, 169.09, 83.24, 82.13, 78.35, 68.84,
68.56, 67.06, 57.51, 52.25, 34.66, 33.45, 28.09 (3C), and 13.85. MS (ESI): m/z
340.3 [M + H]+.
tert-Butyl (S)-3-(2-((3R,5S)-1-pent-4-ynoyl-5-((pyridin-2-ylamino)methyl)
pyrrolidin-3-yloxy)acetamido)-2-(2,4,6-trimethylbenzamido)propanoate
(PMt, 7)
6 (0.11 g, 0.22 mmol) and Et3N (0.46 mL) were dissolved in 4.6 mL of DCM/
dimethyl sulfoxide (DMSO) (75:25) and cooled at 0°C. SO3-pyridine
complex (0.43 g, 2.67 mmol) was added, and the reaction mixture was
stirred for 2 h. The DCM fraction was removed under reduced pressure.
The remaining DMSO fraction was diluted in 30 mL of ethyl acetate.
The organic layer was washed with H2O (30 ml) and saturated NaHCO3
solution (2 × 20 mL). The organic layer was dried over Na2SO4 and
filtered, and the solvent was removed under reduced pressure. The
tert-Butyl 2-((3R,5S)-5-(hydroxymethyl)-1-(pent-4-ynoyl)pyrrolidin-3-yloxy)
acetate (4)
To a cooled solution of 3 (1.15 g, 3.39 mmol) in anhydrous THF/EtOH crude obtained (0.14mg, 0.26 mmol), 2-aminopyridine (0.24 g, 2.58 mmol),
(65:35, 26 mL) at 0°C, LiCl (0.80 g, 18.87 mmol) and NaBH4 (0.30 g,
7.93 mmol) were added. After 1 h, the ice bath was removed, and the
reaction mixture was stirred overnight. Then, the reaction mixture was
and Ti(OiPr)4 (0.96mL, 4.96 mmol) were dissolved in 1.8 mL of
1,2-dichloroethane. After stirring overnight at room temperature, NaBH
(OAc)3 (1.5g, 0.71 mmol) was added, and the reaction was stirred for 4
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J. Label Compd. Radiopharm 2014

A. Monaco et al.
1-Azido-2-[18F]fluoroethane ([18F]12)
additional hours. Saturated NaHCO3 solution (2.70 mL) was added, and the
mixture was stirred for 1 h. It was then extracted with DCM, dried with
Na2SO4, and the solvent removed by evaporation. Light brown oil was
obtained in 67% yield. 1H-NMR (300 MHz, CDCl3) δ: 8.09–8.02 (2H, m),
7.40 (1H, dd, Jd = 1.8, Jd = 8.1Hz), 7.35–7.30 (2H, t, J = 7.8Hz), 6.80 (1H, s),
6.63–6.53 (2H, m), 6.44 (2H, td, Jt = 8.4, Jd = 16.5 Hz), 6.01 (1H, bs), 5.09–
4.99 (1H, m), 4.90–4.78 (1H, m), 4.71–4.52 (2H, m), 4.28 (1H, t, J= 4.5Hz),
4.05–3.58 (5H, m), 2.49–1.94 (13H, m), 1.47 (2H, s), 1.28–1.19 (6H, m), and
0.87 (1H, bs). 13C-NMR (300 MHz, CDCl3) δ: 172.14, 171.10, 169.57, 168.78,
158.28, 157.79, 157.44, 147.79, 137.64, 133.96 (2C), 128.15 (2C), 113.79,
108.53, 83.27, 83.09, 78.12, 77.34, 68.76, 68.17, 62.95, 60.57, 52.79, 42.09,
33.64, 30.79, 27.82 (3C), 20.95, 18.95 (2C), and 13.96. HRMS (ESI) for
The produced carrier-free [18F]fluoride was trapped on a single use light-
QMA anion-exchange column (Waters, Baden-Dättwil, Switzerland), and
the excess of [18O]water was removed. Trapped [18F]fluoride was eluted
from the column with 2.5 mL of a CH3CN/H2O solution (6:1) of K2CO3
(3.5 mg, 25 μmol) and Kryptofix [2.2.2] (K222; 20 mg, 53 μmol) and
transferred into the reactor. This solution was then dried by consecutive
heating at 85 and 110°C under a stream of argon. After cooling the
reactor to 40°C, a solution of 2-azidoethyl-4-methylbenzenesulfonate
(13) (2 mg, 9 μmol) in CH3CN (350 μL) was added to the dry K[18F]F-/
K222 complex, and the reaction mixture was stirred at 80°C for 15 min.
Then, 1-azido-2-[18F]fluoroethane ([18F]12) was purified by distillation at
130°C for 5 min and collected into a vial containing 150 μL of CH3CN.
The radiochemical purity of this intermediate was estimated to be above
98% (Figure 1), and the radiochemical yield was 64% (decay corrected).
C
34H43N5O6 [M+ H]+: calculated m/z 618.3286 and found m/z 618.3282.
tert-Butyl (S)-3-(2-((3R,5S)-1-(3-(1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)
propanoyl)-5-((pyridin-2-ylamino)methyl)pyrrolidin-3-yloxy)acetamido)-
2-(2,4,6-trimethylbenzamido)propanoate (FPMt, 8)
tert-Butyl (S)-3-(2-((3R,5S)-1-(3-(1-(2-[18F]fluoroethyl)-1H-1,2,3-triazol-
4-yl)propanoyl)-5-((pyridin-2-ylamino)methyl)pyrrolidin-3-yloxy)
acetamido)-2-(2,4,6-trimethylbenzamido)propanoate ([18F]FPMt, [18F]8)
7 (10 mg, 0.16 mmol) was dissolved in 1 mL of water/THF, and 12 (62%
m/m in DMF, 20 μL, 7.60 mmol) was added. Sodium ascorbate (0.5 mL,
2.1 M) and CuSO4 (0.5 mL, 0.7 M) were slowly added, and the reaction
was stirred at room temperature for 2 h. The crude was filtered, and
the product was purified by C-18 chromatography to obtain a light
brown solid. Yield: 45%. 1H-NMR (300 MHz, CDCl3) δ: 9.33 (1H, d,
J = 3.9 Hz), 7.75–7.63 (3H, m), 6.82 (2H, s), 4.92 (1H, t, J = 4.7 Hz), 4.76–
4.68 (4H, m), 4.64–4.21 (2H, m), 4.01–3.59 (7H, m), 2.96–2.70 (7H, m),
2.24 (9H, d, J = 5.7 Hz), 1.48 (9H, s), and 0.88 (2H, t, J = 5.6 Hz). 13C-NMR
(300 MHz, CDCl3) δ: 171.35, 169.75, 169.41, 168.76, 161.61, 155.39,
149.30, 146.83, 138.85, 134.06 (2C), 133.42, 128.30 (2C), 126.55, 116.39,
102.89, 83.52, 81.51 (d, J = 171.3 Hz), 80.39, 78.82, 68.24, 62.86, 52.94,
52.64, 50.26, 42.55, 33.06, 31.23, 29.68, 27.95 (3C), 21.07, and 19.04 (2C).
MS (APPI+) for C36H47FN8O6: m/z 707.00 [M + H]+.
Aliquots (50 μL) of the previous solution of 1-azido-2-[18F]fluoroethane
([18F]12) were treated with tert-butyl (S)-3-(2-((3R,5S)-1-pent-4-ynoyl-5-
((pyridin-2-ylamino)methyl)pyrrolidin-3-yloxy)acetamido)-2-(2,4,6-trimethyl
benzamido)propanoate (7) (3mg, 4.85 μmol or 6mg, 9.70 μmol), CuSO4
(50 μL, 0.35M or 1.05 M), and sodium ascorbate (50μL, 0.7 M or 2.1 M) in
a mixture of CH3CN/H2O (1:1, v/v). The reaction mixtures were stirred for
15 min at room temperature and filtered, and then, [18F]8 was purified
by HPLC under the conditions previously described (Figure 2).
Radiochemical yields between 80% and 83% (decay corrected) were
obtained for the click chemistry reactions. Typically, [18F]8 was prepared
in 60–70 min from the end of bombardment (EOB), and the radiochemical
yield was estimated to be 42–44% (decay corrected). Identity of the final
product was confirmed by comparing its HPLC mobility with the retention
time of the nonradioactive standard under the same analytical conditions
used to characterize [18F]12. [18F]8 and 8 had similar retention time of 13.2
and 13.1min, respectively, versus 9.7 and 11.3min for the starting
2-Fluoroethyl 4-methylbenzenesulfonate (11)
Fluoroethanol (10) (1.00 mL, 30.25 mmol) was stirred overnight with p-
toluenesulfonyl chloride (4.80 g, 51.20 mmol) and 7 mL of Et3N in 15 mL
of DCM. The solvent was removed under reduced pressure. Then,
20 mL of EtOAc was added and the mixture extracted with saturated
solution of NaHCO3 (2 × 20 mL). The organic layer was dried over Na2SO4
and filtered, and the solvent was removed under reduced pressure. The
crude obtained was purified by column chromatography on silica gel
using ethyl acetate/hexane (1:5). Brown oil was obtained with a yield of
78%. 1H-NMR (300 MHz, CDCl3) δ: 7.79 (2H, d, J = 8.1 Hz), 7.35 (2H, dd, Jd =
3.9, Jd = 8.4 Hz), 4.13 (2H, td. Jt = 2, Jd = 5.1 Hz), 3.46 (2H, t, J = 4.95 Hz), and
2.43 (3H, s). 13C-NMR (300 MHz, CDCl3) δ: 145.19, 132.32, 129.89 (2C),
127.81 (2C), 68.07, 49.43, and 21.56. MS (ES+): m/z 219 [M + H]+.
materials
[ 7 under the same elution conditions. The
18F]12 and
radiochemical purity of [18F]8 was estimated to be above 95%, and its
specific activity, determined by using on-line measurements of
radioactivity and UV absorption, was >1.3 GBq/μmol.
Results and discussion
Chemistry
Based on docking studies, a pyrrolidine pharmacophore has
been reported to yield selective ligands for integrin α5β1. Indeed,
the 2-aminopyridine and 2,4,6-trimethyl benzoic acid moieties
1-Azido-2-fluoroethane (12)
11 (1.52 g, 7.09 mmol) was dissolved in 3.5 mL of DMF, and NaN3 (1.34 g,
20.60 mmol) was added. The mixture was stirred overnight at 80°C. The
product was purified by azeotropic distillation giving a DMF solution of
12 (62% m/m). 1H-NMR (300 MHz, CDCl3, in DMF solution) δ: 4.56 (2H,
dt, Jt = 4.2, Jd = 38.1 Hz) and 3.49 (2H, d, J = 27.9 Hz). 13C-NMR (300 MHz,
CDCl3, extrapolated form a DMF solution) δ: 99.90 and 82.14 (d,
J = 170 Hz). HRMS (ESI): m/z 90.0462 [M + H]+.
Radiochemistry
18F-Fluoride production
No-carrier-added fluorine-18 was produced according to the nuclear
reaction 18O(p,n)18F by irradiation (10 min at 10 μA) of 2 mL of highly
enriched (>97%) [18O]water (Marshall Isotopes, Tel-Aviv, Israel) by a
proton beam using a Cyclone-18/9 cyclotron (IBA, Louvain-la-Neuve,
Belgium). The initial radioactivity of [18F]fluoride was estimated to be
around 5 GBq.
Figure 1. HPLC radiochromatogram of [18F]12.
J. Label Compd. Radiopharm 2014
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A. Monaco et al.
a
structure closely related to the selective α5β1 antagonist
JSM6427.17 A fluorinated substituent is incorporated on the
pyrrolidine nitrogen in order to not interfere with the binding
of this peptidomimetic molecule with α5β1 integrin receptor.
Synthesis of 7 (Scheme 1) was adapted from the synthesis of
JSM6427, as reported by Stragies et al.
An alkynyl function was introduced into compound 2 by
coupling of 4-pentynoic acid to the 4-hydroxyproline methyl
ester (1) to allow subsequent attachment of the radiolabeled
azido synthon to the alkynyl precursor 7 by the copper(II)-
catalyzed Huisgen’s cycloaddition. Conversion of the secondary
alcohol into alcoholate and Williamson ether synthesis with
tert-butyl bromoacetate provided intermediate 3 in 60% yield.
Deprotection and concomitant reduction of the methyl ester in
position 2 with sodium borohydride in the presence of lithium
chloride yielded intermediate 4. Protection of the primary
alcohol of 4 as tert-butyldimethylsilyl ether afforded compound
5 in 82% yield. The tert-butyl group was then selectively
removed with lithium hydroxide in methanol, and the resulting
carboxylic acid underwent an amidation by treatment with
tert-butyl (S)-3-amino-2-(2,4,6-trimethylbenzamido)propanoate (9)
in presence of 4-dimethylaminopyridine and the coupling reagent
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Deprotection in
position 2 occurred simultaneously to afford the primary alcohol
6 in 40% yield. Compound 9 was synthesized in two steps starting
from commercially available asparagine tert-butyl ester as
previously described.17 Treatment of 6 with the SO3-pyridine
complex to oxidize the primary alcohol into aldehyde, followed
Figure 2. HPLC radiochromatogram of [18F]FPMt ([18F]8). No traces of [18F]12 were
found. The peak at 3.4 min corresponds to an unidentified impurity.
divided by an appropriate linker determine the selectivity for α5β1
integrin receptor.21,22 The 2-aminopyridine interacts with the (α5)-
Phe 187 on the (α5)β-propeller domain, while the mesitylene group
enables π–π interaction with (β1)-Tyr 127 placed in a pocket on the
β1 subunit, which is not available on the αvβ3 surface.21 tert-Butyl (S)-
3-(2-((3R,5S)-1-(3-(1-(2-fluoroethyl)-1H-1,2,3-triazol-4-yl)propanoyl)-
5-((pyridin-2-ylamino)methyl)pyrrolidin-3-yloxy)acetamido)-2-(2,4,
6-trimethylbenzamido)propanoate (FPMt, 8) has been identified,
among 61 α5β1 antagonists, as the most appropriate molecule
for the development of a 18F-labeled α5β1 radioligand. FPMt has
Scheme 1. Synthesis of FPMt (8).
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Copyright © 2014 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2014

A. Monaco et al.
Scheme 2. Radiosynthesis of [18F]FPMt ([18F]8).
by the reductive amidation of the aldehyde with 2-aminopyridine determining the affinity and specificity of [18F]FPMt for integrin
in the presence of Ti(OiPr)4 and NaBH(OAc)3, gave the alkynyl α5β1 and demonstrating how useful this imaging agent may be
precursor 7 in 67% yield. Our optimized synthetic approach in assessing angiogenesis in tumors and monitoring therapy.
allowed us to obtain the alkynyl pyrrolidine derivative 7 in seven
steps, whereas the strategy described by Stragies provided similar
analogs in nine steps. Moreover, 7 was obtained with an overall
satisfactory yield of 45%.
Acknowledgements
This study received funding from the Leenaards Foundation.
We gratefully acknowledge the Center of Biomedical Imaging
for providing resources, the assistance of Dr. V. Zoete (screening
of the α5β1 antagonists), Dr. S. Tardy (helpful discussions),
Dr. E. Rivara-Minten (support at the NMR facility), and the Mass
Spectrometry group of the University of Geneva.
1-azido-2-fluoroetanol (12) was synthesized in two steps.
Activation of the hydroxyl group of 2-fluoroethanol (10) by
a tosylation, followed by a nucleophilic substitution with
NaN3, afforded 12, which was recovered by distillation and
obtained as DMF solution (62% m/m of 12) in 42% yield.23,24
Finally, the Cu(II)-catalyzed Huisgen’s 1,3-dipolar cycloaddition
between
7 and the azido synthon 12 gave the final
Conflict of Interest
peptidomimetic molecule 8 (FPMt) in only 2 h with 45%
yield.25 The time of reaction and the yield obtained suggested
that this strategy could be applied for the development of an
18F-labeled analog.
The authors did not report any conflict of interest.
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Conclusions
Herein, we report the synthesis of [18F]FPMt ([18F]8), a nonpeptidic
radiopharmaceutical for PET imaging with a structure derived from
a selective antagonist of α5β1 integrin receptor.17 Our alkynyl
precursor 7 was successfully synthesized, and all the steps were
optimized to provide good yields. 1-azido-2-fluoroethane (12)
was chosen as synthon for the development of the PET tracer,
and it was clicked to 7 following the standard Cu catalyzed
alkyne-azide click reaction. Future studies will be aimed at
J. Label Compd. Radiopharm 2014
Copyright © 2014 John Wiley & Sons, Ltd.
www.jlcr.org

A. Monaco et al.
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www.jlcr.org
Copyright © 2014 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2014