Identification and in vivo evaluation of a fluorine-18 rolipram analogue, [18F]MNI-617, as a radioligand for PDE4 imaging in mammalian brain

Article abstract of DOI:10.1002/jlcr.3389

Phosphodiesterase (PDE) 4 is the most prevalent PDE in the central nervous system (CNS) and catalyzes hydrolysis of intracellular cAMP, a secondary messenger. By therapeutic inhibition of PDE4, intracellular cAMP levels can be stabilized, and the symptoms of psychiatric and neurodegenerative disorders including depression, memory loss and Parkinson's disease can be ameliorated. Radiotracers targeting PDE4 can be used to study PDE4 density and function, and evaluate new PDE4 therapeutics, in vivo in a non-invasive way, as has been shown using the carbon-11 labeled PDE4 inhibitor R-(-)-rolipram. Herein we describe a small series of rolipram analogs that contain fluoro- or iodo-substituents that could be used as fluorine-18 PET or iodine-123 SPECT PDE4 radiotracers. This series was evaluated with an in vitro binding assay and a 4-(fluoromethyl) derivative of rolipram, MNI-617, was identified, with a five-fold increase in affinity for PDE4 (K_d = 0.26 nM) over R-(-)-rolipram (K_d = 1.6 nM). A deutero-analogue d₂-[¹⁸F]MNI-617 was radiolabeled and produced in 23% yield with high (>5 Ci/μmol) specific activity and evaluated in non-human primate, where it rapidly entered the brain, with SUVs between 4 and 5, and with a distribution pattern consistent with that of PDE4.

Full text of DOI:10.1002/jlcr.3389

Research article
Received 4 February 2016,
Revised 18 February 2016,
Accepted 22 February 2016
Published online 22 March 2016 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3389
Identification and in vivo evaluation of a
18
fluorine-18 rolipram analogue, [ F]MNI-617,
as a radioligand for PDE4 imaging in
mammalian brain
*
David Thomae, Thomas J. Morley, Hsiaoju S. Lee, Olivier Barret,
Cristian Constantinescu, Caroline Papin, Ronald M. Baldwin,
Gilles D. Tamagnan, and David Alagille
Phosphodiesterase (PDE) 4 is the most prevalent PDE in the central nervous system (CNS) and catalyzes hydrolysis of
intracellular cAMP, a secondary messenger. By therapeutic inhibition of PDE4, intracellular cAMP levels can be stabilized,
and the symptoms of psychiatric and neurodegenerative disorders including depression, memory loss and Parkinson’s
disease can be ameliorated. Radiotracers targeting PDE4 can be used to study PDE4 density and function, and evaluate
new PDE4 therapeutics, in vivo in a non-invasive way, as has been shown using the carbon-11 labeled PDE4 inhibitor
R-(À)-rolipram. Herein we describe a small series of rolipram analogs that contain fluoro- or iodo-substituents that could
be used as fluorine-18 PET or iodine-123 SPECT PDE4 radiotracers. This series was evaluated with an in vitro binding assay
and a 4-(fluoromethyl) derivative of rolipram, MNI-617, was identified, with a five-fold increase in affinity for PDE4
(Kd = 0.26 nM) over R-(À)-rolipram (Kd = 1.6 nM). A deutero-analogue d2-[18F]MNI-617 was radiolabeled and produced in
23% yield with high (>5 Ci/μmol) specific activity and evaluated in non-human primate, where it rapidly entered the brain,
with SUVs between 4 and 5, and with a distribution pattern consistent with that of PDE4.
Keywords: PET imaging; phosphodiesterase; PDE4; fluorine-18; Parkinson’s disease
density of this enzyme in vivo using a non-invasive technique
Introduction
would be invaluable in studying the role of the enzyme
in vivo.15 Additionally, it can be used to study the target-
The nucleotide cyclic adenosine 3′,5′-monophosphate (cAMP) is an
intracellular secondary messenger for many signal transduction
engagement and efficacy of new PDE4-specific pharmaceutical
pathways. Adenylate cyclase catalyzes the formation of cAMP from
candidates. Indeed, rolipram has been labeled with carbon-11
([11C]1) and successfully used as a radiotracer to quantify PDE4
adenosine 5′-triphosphate (ATP), which is stimulated by activation
of ß-adrenergic, adenosine and histamine receptors.1 Intracellular
levels of cAMP are reduced by hydrolysis of this messenger by
one, or more, of the 11 known families of phosphodiesterase
(PDEs) enzymes.2,3 Dysregulation of cAMP signaling in the
brain has been implicated in several psychiatric,4 and
neurodegenerative disorders including Alzheimer’s disease
(AD),5 Parkinson’s disease (PD)6 and Huntingdon’s disease (HD).7
Of the 11 PDE families, PDE4 is the most abundant in the
central nervous system (CNS), accounting for 30–70% of total
PDE activity, and is widespread throughout the brain.8
Inactivation of PDE4, by specific inhibitors,9 serves to stabilize
intracellular cAMP levels and can improve the symptoms of
these disorders.10,11 Rolipram 1 was the first selective PDE4
inhibitor to be clinically investigated,12,13 and has since been
followed by roflumilast 2 (Scheme 1).14 Both drugs are based
on a catechol core bearing a cyclopentyl and methyl substituent
at the 3- and 4-positions, respectively.
in rat,16 pig,17 and human brain using positron emission
tomography (PET).18 The radiotracer showed good brain uptake
(SUV = 1.5 to 3) and a widespread distribution in all brain regions,
followed by a gradual washout over 90 min. The related tracer
[11C]Ro-20,1724 ([11C]3) has also been prepared and tested in
rats, but was found to have a much lower brain uptake (0.4
versus 1.2 ID/g) compared to [11C]rolipram.19,20
Based on these results we sought to develop new tracers
based on the structure of rolipram, but labeled with the longer
lived radiohalogens fluorine-18 (110 min) and iodine-123
(13.1 h). To this end, a small series of rolipram analogues were
synthesized that contained either fluorine or iodine incorporated
into substituents in the 3- and 4-positions. In vitro testing of this
Molecular NeuroImaging LLC, New Haven, CT 06510, USA
*Correspondence to: Thomas J. Morley, Molecular NeuroImaging LLC, New
Haven, CT 06510, USA.
E-mail: tmorley@mnimaging.com
Because of the prevalence of PDE4 in the CNS and its role in
neurological disease states, tools to study the function and
J. Label Compd. Radiopharm 2016, 59 205–213
Copyright © 2016 John Wiley & Sons, Ltd.

D. Thomae et al.
Scheme 1. Structures of specific PDE4 pharmaceuticals and radiotracers.
Scheme 2. General synthetic route to 3- or 4-substituted R-(À)-rolipram derivatives. a) CH3NO2, AcOH, NH4OAc, reflux, 4 h; b) LiHMDS 1 M in THF, (R)-3-acetyl-4-
benzyloxazolidin-2-one, THF, À78 °C; c) NiCl2.6H2O, NaBH4, 0 °C to RT, 4 h.
Scheme 3. Synthesis of 3- and 4-substituted analogues of R-(À)-rolipram 10a-f and 11a-g. Reagents and conditions: i) TsO(CH2)2F, DMF, K2CO3, 90 °C overnight (65% of
10a and 52% for 11a); ii) NaH, DMF, di-p-tosyl-1,3-propanediol, 3 h, RT; iii) TBAF, THF, reflux 5 h (94% for 10b and 55% for 11b, over two steps); iv) (E)-4-Fluorobut-2-en-1-yl
4-methylbenzenesulfonate, DMF, K2CO3, 90 °C overnight (41% for 10c and 51% for 11c); v) NaH, DMF, 4-Fluorobut-2-yn-1-yl methanesulfonate, 90 °C overnight (50% for
10d and 58% for 10d); vi) NaH, DMF, 2-(2-Fluoroethoxy)ethyl 4-methylbenzenesulfonate, 4 h, RT and 90 °C overnight (35% for 10e and 54% for 11e); vii) NaH, DMF,
fluoromethyl 4-methylbenzenesulfonate, 4 h, RT and 90 °C overnight (13% for 10f and 15% for 11f); viii) (E)-3-(Tributylstannyl)allyl 4-methylbenzenesulfonate,24 DMF,
K2CO3, 90 °C overnight, ix) I2, CH2Cl2 (54% for 11g over two steps).
Scheme 4. Synthesis of 12 and 16: Reagents and conditions: i) CH3NO2, AcOH, NH4OAc, reflux, 4 h (92%); ii) LiHMDS 1 M in THF, (R)-3-acetyl-4-benzyloxazolidin-2-one, THF,
À78 °C, 1 h; iii) Fe, AcOH, THF, HCl 2 M (44% over two steps); iv) hexamethylditin, Pd(Ph3)4, DME, reflux 5 h (18%); v) I2, CH2Cl2 (79%); vi) NaH, DMF, MeI, 2 h at RT (58%); vii)
NaH, DMF, TsO(CH2)2F (52%).
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D. Thomae et al.
Scheme 5. General structure of R-(À)-rolipram derivatives (Table 1) tested in
competition binding assay. The binding affinity of all fluoro- and iodo-containing
rolipram analogs for the PDE4 receptor were evaluated in vitro by CEREP
(Redmond, WA) using a competition assay against [3H]R-(À)-rolipram (at 1 nM)
using mouse brain homogenates.31 These assays were performed with R-(À)-
rolipram as a reference, which had a Kd of 1.6 nM.
Scheme 6. Radiosynthesis of d2-[18Fnn11f (d2-[18F]MNI-617).
was synthesized from 3-benzyloxy-4-methoxybenzaldehyde, and
phenol 9 was synthesized from 3-cyclopentoxy-4-(benzyloxy)
series in a competition binding assay with [3H]rolipram identified benzaldehyde (Scheme 3). O-Alkylation of the sodium salt of
a candidate for a PDE4 PET radiotracer, MNI-617. This candidate phenols 8 or 9 with the corresponding alkyl tosylates gave
was radiolabeled with [18F]fluoride in a commercial automated derivatives 10a–f and 11a–g.25–28
module and evaluated in vivo by PET imaging in non-human
primates.
A 3-iodo analogue of rolipram 12 was synthesized as shown in
Scheme 4, which follows a similar sequence to that shown in
Scheme 2, but starts with 3-bromo-4-methoxybenzaldehyde 13.
In this sequence the reduction of nitrovinyl 14 step was performed
with iron in hydrochloric acid to avoid dehalogenation,29 which
Results and discussion
The rolipram analogues were synthesized by replacing the 4- gave lactam 15 in 40% over three steps. Coupling of 15 with
methoxy and the 3-cyclopentoxy substituents of rolipram with hexamethylditin gave a trimethylstannane intermediate,30 which
groups bearing either a fluorine or iodine atom, which would was treated with iodine to give desired 3-iodo derivative 12.
allow for easy incorporation of the radiohalogens fluorine-18 or R-(À)-Rolipram 1 itself was synthesized by alkylation of phenol
iodine-123 into the final radiotracer. The general synthetic route 9 with bromocyclopentane for use as reference for the in-vitro
to these compounds follows a previously published route and is binding tests. Finally, an N-substituted derivative of R-(À)-
shown in Scheme 2.21
rolipram 16 was synthesized by alkylation of 1 with 1,2-
This route starts with the Henry reaction of a 3,4-disubsituted fluoroethyltosylate.
benzaldehyde, which contains the desired substituents at 3- and
From the results of these in vitro binding assays in Table 1, it is
4-positions 4, with nitromethane to give nitrovinyl 5. Next, a clear that structural modifications to R-(À)-rolipram (Scheme 5)
Michael reaction using lithiated (R)-3-acetyl-4-benzyloxazolidin- are not well tolerated by the PDE4 receptor, with large losses
2-one22 gives intermediate 6 as a mixture of the (R,R) and (R,S) (>50-fold increase in Kd) in binding affinity for eight out of
diastereoisomers in a 94 to 6 ratio. This mixture of isomers is the 15 compounds. Any modification to the 3-position resulted
used without further purification by reducing the nitro group in an increase in Kd compared to R-(À)-rolipram, with the best
under mild conditions,23 with the resulting amine cyclizing in- compounds of the series, the fluoroalkyl derivatives 10a, 10b
situ to give lactam 7 over two steps. Finally, any protecting and 10c possessing a Kd of 6.8, 2.4 and 13 nM, respectively.
groups are removed to give the desired rolipram analogue.
Similarly, increasing the size of the group at the 4-position
For the synthesis of 3- or 4-O-alkyl derivatives of R-(À)-rolipram, above that of a fluoroethyl group (Kd = 9.8 nM for 11a) resulted
phenols 8 and 9, respectively, were required. These were in almost no affinity for the PDE4 receptor (Kd > 1000 nM),
synthesized using the route shown in Scheme 2, where phenol 8 except fluoromethyl derivative 11f, which had an increased
Table 1. PDE4 binding affinity of analogs of R-(À)-rolipram, displacing [3H]R-(À)-rolipram
#
R1
R2
R3
Kd (nM)
clogD
1
Me
Me
Me
Me
Me
Me
Me
O
cycPent
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
1.6
6.3
2.4
13
100
33
250
9.8
0.80
1.41
1.64
1.73
2.16
1.16
1.33
2.09
2.32
2.42
2.84
1.84
2.02
3.39
2.40
2.63
10a
10b
10c
10d
10e
10f
11a
11b
11c
11d
11e
11f
11g
13
OCH2CH2F
OCH2CH2CH2F
(E)-OCH2CHCHCH2F
OCH2CCCH2F
OCH2CH2OCH2CH2F
OCH2F
CH2CH2F
OcycPent
CH2CH2CH2F
(E)-CH2CHCHCH2F
CH2CCCH2F
CH2CH2OCH2CH2F
CH2F
(E)-CH2CHCHI
Me
Me
OcycPent
>1000
>1000
210
OcycPent
OcycPent
OcycPent
>1000
OcycPent
0.26
110
74
OcycPent
I
16
OcycPent
CH2CH2F
8.3
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D. Thomae et al.
Figure 1. (A) Images of [18F]MNI-617 accumulation in rhesus brain over 2 h following intravenous injection and (B) decay-corrected time-SUV curves in several regions of
the brain.
affinity, to 0.26 nM. The N-fluoroethyl derivative 16 also 120 °C for 10 min. The crude product mixture was purified by
showed a decrease in binding affinity to 8.3 nM, making semi-preparative reverse-phased HPLC, formulated into normal
fluoromethyl derivative 11f the most suitable candidate of this saline containing ascorbic acid, and filter-sterilized into a sterile
series for [18F]fluorination and development into a PET radiotracer. final product vial. The resulting formulated product had a
The two iodo-compounds 11g and 13 both showed a marked radiochemical purity of 99.9% with a specific activity of
decrease in binding from R-(À)-rolipram, to 110 and 74 nM 348 GBq/μmol (9.4 Ci/μmol) and was obtained in
a 23%
respectively, making them unsuitable for development into SPECT decay-corrected radiochemical yield (from [18F]fluoride) in a
radiotracers.
production time of 85 min (n = 3). d2-[18F]11f had a measured
Although 11f was chosen for further development on binding logD of 1.9, using the shake-flask method.34
affinity for PDE4 alone, this series of compounds was not
screened against other CNS or PDE targets, which could affect
their suitability as a PDE4-specific radiotracer.
In vivo imaging
[
18F]11f was evaluated in vivo in an 11.3 kg anesthetized female
Radiochemistry
rhesus monkey. After 4.5–7 min following bolus injection of the
Compound 11f, once radiolabeled, would carry the radiohalogen tracer (4.8–5.0 mCi, 0.4–0.7 μg, 140–350 GBq/μmol, n = 2), high
as [18F]fluoromethyl-group, and this moiety is known to be brain uptake was observed, with SUVs of 2.9 in the cerebellum,
susceptible to defluorination in vivo, leading to bone uptake of free 3.1 in the putamen, 3.4 in the anterior cingulate and 3.7 in the
[18F]fluoride. One of the commonly used methods to increase thalamus, as shown in Figure 1. This pattern is consistent
metabolic stability of these groups and reduce defluorination is with the known distribution of PDE4 throughout the brain.8
to use the deuterium analogue d2-fluoromethyl ([18F]FCD2) d2-[18F]11f also showed fast kinetics, with rapid accumulation
instead.32 The deuterium substitution should have a negligible and washout. Binding potentials (BPND) calculated using non-
effect on the binding affinity and it is for this reason that we chose invasive Logan graphical analysis with cerebellar vermis as
to synthesize the deuterated analogue of 11f for in vivo testing.
reference region (low PDE4) ranged between 0.3 (frontal
The fluorine-18 derivative d2-[18F]11f (d2-[18F]MNI-617) was cortex, anterior cingulate) and 0.7 (thalamus). Although these
synthesized on a GE TRACERlab® FXF-N automated synthesis BPND values are low, they are comparable with similar values
module using a two-step method (Scheme 6). The first step measured for R-(À)-[11C]rolipram.35,36 The radiotracer was
was production of d2-[18F]fluoromethyl tosylate by [18F] rapidly metabolized in vivo, with less than 5% of the parent
fluorination of d2-methylene bis-tosylate in the presence of d2-[18F]11f remaining in the plasma after 15 min. The metabolism
cryptand-222 (K222) and potassium carbonate.33 The resulting was accompanied by an accumulation of radioactivity in the skull,
d2-[18F]fluoromethyl tosylate was added to a solution of phenol indicating [18F]fluoride uptake from defluorination of the
9 in DMSO in the presence of cesium carbonate and heated to radiotracer.
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D. Thomae et al.
To assess the specificity of d2-[18F]11f for PDE4, a single
blockade experiment was carried out in the same animal by
treatment with 50 μg/kg of R-(À)-rolipram, administered as
5 min infusion at 30 min before injection of the radiotracer.
Although a faster washout of d2-[18F]11f was observed at
blockade than at baseline in the selected brain regions, the
rapid metabolism of the radiotracer meant that invasive
quantification (with arterial blood) could not be performed,
and no conclusive blockade result could be confirmed.
Sigma, MO). Cryptand 222 (Kryptofix-222) content was measured using a
TLC test as published.37 Residual solvent analysis was performed using a
Perkin-Elmer Clarus 500 gas chromatograph fitted with a Restek Stabilwax
(30 m × 0.53 mm × 1 μm) column. Pyrogen content was measured by the
LAL test with a Charles River Laboratories Endosafe PTS-100. Sterility tests
were performed by inoculating a sample of the drug product in fluid
thioglycollate medium and tripticase soy broth (Northeast Laboratories)
and monitoring for two weeks, per USP guidelines.
In vivo imaging studies were performed at the Yale University School
of Medicine in New Haven, Connecticut using a female rhesus monkey
(Macaca mulatta). PET imaging was carried out under institutional animal
care protocols complying with Federal regulations. Animal care approval
and oversight for this study were provided by the Yale University
Institutional Animal Care and Use Committee. The animal was fasted
for up to 12 h before imaging. At 2 h prior to injection of the radiotracer,
the animal was anesthetized with intramuscular ketamine (10 mg/kg),
treated with glycopyrrolate (0.1–0.2 mg/kg), transferred to the PET
camera and immediately intubated with an endotracheal tube for
continued anesthesia with 1.5–2.5% isoflurane administered through a
re-breathing circuit. An intravenous (i.v.) line was used for injection of
fluids for hydration and radiotracer injection. Body temperature was kept
Conclusion
We have synthesized 15 analogs of R-(À)-rolipram, which
contain fluoro- or iodo-substituents, and evaluated them
in vitro against R-(À)-rolipram itself. This screen provided
MNI-617 (11f) as a potential PET radioligand, with a Kd of
0.26 nM, a five-fold improvement over rolipram. A deuterated
analogue of 11f, d2-[18F]11f ([18F]MNI-617) was synthesized
using a two-step method on a commercial GE TRACERlab
FXFN with high specific activity and radiochemical purity and at approximately 37 °C using a heated water blanket. Vital signs,
including heart rate, blood pressure, respiration rate, oxygen saturation
and body temperature, were monitored at least every 15 min during
the study. The animal received two scans, at baseline and following
pre-injection of the PDE4 inhibitor R-(À)-rolipram. d2-[18F]11f was
administered iv as a bolus over 3 min. List mode emission data was
collected over 2 h on the microPET FOCUS 220 camera (Siemens Medical
Solutions, Inc.). A transmission scan with a Ge-68 source prior to injection
of radiotracer. Data was reconstructed into a series of 33 dynamic 3D
images (6 × 0.5, 3 × 1, 2 × 2 and 22 × 5 min) using filtered back
projection. Corrections for random, dead time, scatter, attenuation,
and decay were applied.
evaluated as a potential PET radiotracer. PET imaging of d2-
18F]11f in rhesus monkey demonstrated high brain uptake,
with SUVs of 3–4, with similar binding potentials to R-(À)-
11C]rolipram, making it an attractive alternative PET tracer
for imaging of PDE4 in brain. However, modifications to
overcome the rapid metabolism as observed by in vivo de-
fluorination and further displacement or blockade studies
are required to demonstrate that this radiotracer has
acceptable in vivo properties and specificity for PDE4.
[
[
General Method for Alkylation of Phenols—To a solution (50 mM) of
the phenol (1.0 eq.) in DMF was added alkylating agent (1.2 eq.) and
potassium carbonate (3.0 eq.). The mixture was heated for the stated
time and temperature and after cooling to room temperature water
(10 mL) was added and the mixture extracted with ethyl acetate
(3 × 10 mL). The combined organic layers were washed with water
(2 × 10 mL), dried (Na2SO4) and evaporated under reduced pressure.
The residue was purified by flash column chromatography.
Experimental
Commercially available reagents were purchased from Aldrich, and used
as supplied without further purification. Reactions were carried out under
an atmosphere of nitrogen. Analytical thin layer chromatography was
carried out using aluminum-backed plates coated with Merck Kieselgel
60 GF254. Visualization was realized by UV light (254 nm) or with a
ninhydrin stain. Flash chromatography was performed using silica gel
(60 Å, 200–425 mesh). 1H-NMR and 13C-NMR spectra were recorded on
an AC Bruker 500 MHz instrument. All NMR spectra were recorded at
23 °C. Proton and carbon chemical shifts (δ) are expressed in parts per
million (ppm) downfield from tetramethylsilane and are referenced to
the atom resonance of the solvent (CHCl3: δ = 7.26 ppm from 1H and
δ = 77.2 for 13C). For 1H NMR, data are presented as follows: chemical
shift, multiplicity (s, singlet; d, doublet; dt, doublet of triplet; m, multiplet;
(R)-4-(3-(2-fluoroethoxy-4-methoxyphenyl)pyrrolidin-2-one (10a)
Phenol 8 (150 mg, 0.347 mmol) in DMF (7 mL) was combined with
2-fluoroethyl 4-methylbenzenesulfonate (220 mg, 1.6 mmol) and potassium
carbonate (349 mg, 1.6 mmol) at 90 °C for 18 h according to General
Methods. The residue was purified (SiO2: ethyl acetate/hexanes 8/2 then
dichloromethane/methanol 17:3) to obtain pyrrolidinone 10a (120 mg,
65%) as a white solid: 1H-NMR (500 MHz, CDCl3): δH = 7.01 (bs, 1H), 6.85 (m,
3H), 4.77 (dd, 2H, J = 47.5 Hz and J = 4.2 Hz), 4.28 (dd, 2H, J = 27.9 Hz and
J = 4.2 Hz), 3.86 (s, 3H), 3.76 (t, 1H, J = 9.4 Hz), 3.62 (quintet, 1H, J = 8.9 Hz),
3.38 (dd, 1H, J = 9.2 Hz and 7.6 Hz), 2.70 (dd, 1H, J = 16.9 Hz and 9.0 Hz), 2.47
(dd, 2H, J = 16.9 Hz and J = 9.0 Hz). 13C-NMR (125 MHz, CDCl3): δC = 178.33 (C),
148.47 (C), 149.38 (C), 135.24 (C), 120.58 (CH), 113.99 (CH), 112.81 (CH), 82.45
(d, CH2, J = 169.6 Hz), 69.22 (d, CH2, J = 20.3 Hz), 56.47 (CH3), 50.18 (CH2),
40.24 (CH), 36.63 (CH2). HRMS ES(+): m/z calcd for C13H17FNO3 (M + H)+:
254.1192, found: 254.1189. Elemental Analysis: calcd: C: 61.65; H: 6.37, N: 5.53;
found: C: 61.56; H: 6.42; N: 5.41. α2d0 (CHCl3, C = 0.25): À22.0°.
bs, broad signal), coupling constants (J in Hertz) and integration. For 13
C
NMR data, signals corresponding to CH, CH2 or CH3 groups are assigned
from DEPT. High Resolution Mass Spectroscopy (HRMS) was recorded at
the School of Chemical Science, University of Illinois, with an electron
ionization spray (ESI) technique. All compounds tested were >95% pure
by elemental and/or HPLC analysis. Elemental analyses were performed
by Atlantic Microlab (Norcross, GA).
In vitro binding assays were performed by CEREP (Redmond, WA)
using a competition assay against [3H]R-(À)-rolipram (at 1 nM) using
mouse brain homogenates.31
Radiolabeling was performed on a GE TRACERlab FXFN module using
helium pressure or vacuum for fluid transfers.
purchased from Cardinal Health (Hartford, CT). QC HPLC testing was
[
18F]Fluoride was
(R)-4-(3-(3-fluoropropoxy)-4-methoxyphenyl)pyrrolidin-2-one (10b)
performed with a Waters HPLC system (1525 or e2695), monitoring the To a cooled (0 °C) solution of phenol 8 (207 mg, 1.0 mmol) in DMF (5 mL)
column effluent with ultraviolet (Waters 2489, λ = 280 nm) and was added sodium hydride (60% dispersion in oil, 44 mg, 1.1 mmol). After
radioactivity detectors (Bioscan FC-200) with the data analyzed with
stirring for 20 min the mixture was added to a solution of 1,3-bis-
Waters Empower software. Radioactivity was measured with a Capintec (4-methylbenzenesulfonate)-propane (1.04 g, 3.0 mmol) in DMF (3 mL)
CRC-25 dose calibrator. Radionuclidic identity was determined by
gamma spectroscopy using a Canberra 802 multi-channel analyzer. pH
measurements were performed with pH indicator strips (Range 0–14,
and allowed to warm to room temperature over 3 h. Water (10 mL) was
added and the mixture extracted with ethyl acetate (3 × 10 mL). The
combined organic layers were washed with water (2 × 5 mL), dried
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D. Thomae et al.
(Na2SO4) and evaporated under reduced pressure. The residue was
purified by flash column chromatography (SiO2: ethyl acetate/hexanes
8/2) to obtain (R)-3-(2-methoxy-5-(5-oxopyrrolidin-3-yl)phenoxy)propyl
4-methylbenzenesulfonate (290 mg, 70%) as a colorless oil. This tosyl
intermediate (230 mg, 0.56 mmol) was dissolved in THF (5 mL), and TBAF
solution (1 M in THF, 2.8 mL, 2.8 mmol) was added and the mixture heated
to reflux for 5 h. After cooling to room temperature the excess solvent was
evaporated under reduced pressure. The residue was dissolved in ethyl
acetate (30 mL) and washed with diluted hydrochloric acid (2 M, 10 mL).
The organic phase was dried (Na2SO4), evaporated under reduced pressure
and the residue was purified by flash column chromatography (SiO2:
dichloromethane/methanol 95:5) to obtain pyrrolidinone 10b (60 mg,
44%) as a white solid. 1H-NMR (500 MHz, CDCl3): δH = 6.82–6.88 (m, 3H),
5.52 (bs, 1H), 4.70 (dt, 2H, J = 47.1 Hz and J = 5.7 Hz), 4.18 (t, 2H, J = 6.3 Hz),
3.88 (s, 3H), 3.78 (t, 1H, J = 9.2 Hz), 3.67 (quintet, 1H, J = 8.5 Hz), 3.40 (t, 1H,
J = 7.5 Hz), 2.74 (dd, 1H, J = 17.0 Hz and 9.0 Hz), 2.50 (dd, 1H, J = 17.0 Hz
and 9.0 Hz), 2.25 (dq, 2H, J = 26.4 Hz and J = 6.1 Hz). 13C-NMR (125 MHz,
CDCl3): δC = 178.47 (C), 149.04 (C), 148.85 (C), 135.22 (C), 119.76 (CH),
112.81 (CH), 112.48 (CH), 81.20 (d, CH2, J = 163.2 Hz), 65.39 (d, CH2,
J = 4.8 Hz), 56.47 (CH3), 50.24 (CH2), 40.30 (CH), 38.70 (CH2), 30.85 (d, CH2,
J = 19.8 Hz). HRMS ES(+): m/z calcd for C14H19FNO3 (M + H)+: 268.1349,
found: 268.1342. Elemental Analysis: calcd: C: 62.91; H: 6.79, N: 5.24; found:
C: 62.95; H: 6.76; N: 5.18. α2d0 (CHCl3, C = 0.25): À25.0°.
(R)-4-(3-(2-(2-fluoroethoxy)ethoxy)-4-methoxyphenyl)pyrrolidin-2-
one (10e)—
To a cooled (0 °C) solution of phenol 8 (200 mg, 0.97 mmol) in DMF
(5 mL) was added sodium hydride (60% dispersion in oil, 43 mg,
1.06 mmol). After stirring for 20 min the mixture was added to a
solution
of
2-(2-fluoroethoxy)ethyl
4-methylbenzenesulfonate
(277 mg, 1.06 mmol) in DMF (1 mL). The mixture was allowed to
warm to room temperature over 4 h and then heated to 90 °C for
18 h. After cooling to room temperature ware (10 mL) was added
and the mixture was extracted with ethyl acetate (3 × 10 mL). The
combined organic layers were washed with water (2 × 5 mL), dried
(Na2SO4) and evaporated under reduced pressure. The residue was
purified by flash column chromatography (SiO2: dichloromethane/
methanol 9/1) and recrystallized with ethyl acetate and hexanes to
give pyrrolidinone 10e (100 mg, 35%) as a white solid: 1H-NMR
(500 MHz, CDCl3): δH 6.83 (m, 4H), 4.58 (dt, 2H, J = 47.7 Hz and
8.9 Hz ), 4.21 (m, 2H), 3.91 (m, 2H), 3.85 (m, 4H), 3.79 (m, 1H), 3.75
(m, 1H), 3.62 (m, 1H), 3.38 (m, 1H), 2.70 (dd, 1H, J = 16.9 Hz and
8.9 Hz), 2.46 (dd, 1H, J = 16.9 Hz and 8.9 Hz). 13C-NMR (125 MHz,
CDCl3): δC = 178.25 (C), 149.23 (C), 148.84 (C), 135.18 (C), 120.08
(CH), 113.64 (CH), 112.50 (CH), 83.56 (d, CH2, J = 169 Hz), 70.99 (d,
CH2, J = 20 Hz), 70.37 (CH2), 69.42 (CH2), 56.44 (CH3), 50.12 (CH2),
40.30 (CH), 38.57 (CH2). HRMS ES(+): m/z calcd for C15H21NO4F (M
+ H)+: 298.1455, found: 298.1455. Elemental Analysis: calcd: C: 60.59;
H: 6.78; N: 4.70.; found: C: 60.31; H: 6.74; N: 4.70. α2d0 (CHCl3,
C = 0.17): À3.4°.
(R,E)-4-(3-((4-fluorobut-2-en-1-yl)oxy)-4-methoxyphenyl)pyrrolidin-
2-one (10c)
To a cooled solution of phenol 8 (200 mg, 0.97 mmol) in DMF (5 mL)
was added sodium hydride (60% dispersion in oil, 43 mg, 1.06 mmol).
(R)-4-(3-(fluoromethoxy)-4-methoxyphenyl)pyrrolidin-2-one (10f)—
After stirring for 20 min
a solution of 2-((E)-4-fluoro-2-butenyl)-
To a cooled (0 °C) solution of phenol 8 (200 mg, 0.97 mmol) in DMF (5 mL)
was added sodium hydride (60% dispersion in oil, 43 mg, 1.06 mmol).
After stirring for 20 min the mixture was added to a solution of
fluoromethyl 4-methylbenzenesulfonate (216 mg, 1.06 mmol) in DMF
(1 mL). The mixture was allowed to warm to room temperature over 4 h
and then heated to 90 °C for 18 h. After cooling to room temperature
ware (10 mL) was added and the mixture was extracted with ethyl
acetate (3 × 10 mL). The combined organic layers were washed with
water (2 × 5 mL), dried (Na2SO4) and evaporated under reduced pressure.
The residue was purified by flash column chromatography (SiO2, ethyl
acetate/hexanes, 8/2 then ethyl acetate/methanol, 9/1) to obtain
pyrrolidinone 10f (40 mg, 13%) as a white solid: 1H-NMR (500 MHz,
CDCl3): δH 7.35 (bs, 1H), 7.01 (s, 1H), 6.96 (d, 1H, J = 8.4 Hz), 6.87 (d, 1H,
J = 8.4 Hz), 5.68 (d, 1H, J = 54.6 Hz), 3.83 (s, 3H), 3.75 (t, 1H, J = 8.6 Hz),
3.61 (m, 1H), 3.37 (t, 1H, J = 8.4 Hz), 2.70 (dd, 1H, J = 16.8 Hz and 8.9 Hz),
2.45 (dd, 1H, J = 16.7 Hz and 8.9 Hz). 13C-NMR (125 MHz, CDCl3):
δC = 178.54 (C), 149.26 (C), 146.10 (C), 135.29 (C), 122.86 (CH), 117.20
(CH), 112.89 (CH), 102.05 (d, CH2, J = 218.1 Hz), 56.41 (CH3), 50.24
(CH2), 40.00 (CH), 38.65 (CH2). Elemental Analysis: calcd: C: 60.24; H:
5.90, N: 5.85; found: C: 60.35; H: 5.91; N: 5.94. HRMS ES(+): m/z calcd
4-methylbenzenesulfonate (282 mg, 1.16 mmol) in DMF (3 mL) was
added, and the solution was allowed to warm to room temperature
over 2 h. Water (10 mL) was added, and the mixture was extracted
with ethyl acetate (3 × 5 mL). The combined organic layers were
washed with water (2 × 5 mL), dried (Na2SO4) and evaporated under
reduced pressure. The residue was purified by flash column
chromatography (SiO2: dichloromethane/methanol 9/1) and recrystallized
from ethyl acetate and hexanes to give pyrrolidinone 10c (110 mg, 41%)
1
as a white solid. H-NMR (500 MHz, CDCl3): δH 6.82–6.87 (m, 2H), 6.78 (m,
1H), 6.43 (bs, 1H), 6.06 (m, 2H), 4.91 (m, 2H), 4.65 (m, 2H), 3.88 (m, 3H),
3.77 (m, 1H), 3.64 (m, 1H), 3.39 (m, 1H), 2.72 (dd, 1H, J = 16.8 Hz and
8.9 Hz), 2.47 (dd, 1H, J = 16.9 Hz and 8.6 Hz). 13C-NMR (125 MHz, CDCl3):
δC = 178.04 (C), 149.05 (C), 148.40 (C), 135.13 (C), 129.71 (d, CH,
J = 11.6 Hz), 128.46 (d, CH, J = 16.8 Hz), 119.89 (CH), 112.81 (CH), 112.30
(CH), 83.87 (d, CH2, J = 161.3 Hz), 69.03 (CH2), 56.43 (CH3), 50.09 (CH2),
40.32 (CH), 38.47 (CH2). HRMS ES(+): m/z calcd for C15H19 FNO3 (M + H)+:
280.1349, found: 280.1350. Elemental Analysis: calcd: C: 64.50; H: 6.50, N:
5.01; found: C: 64.37; H: 6.34; N: 4.94. α2d0 (CHCl3, C = 0.5): À12.0°.
for
C
12H15FNO3 (M + H)+: 240.1036, found: 240.1028. α2d0 (MeOH,
(R)-4-(3-((4-fluorobut-2-yn-1-yl)oxy)-4-methoxyphenyl)pyrrolidin-2-
one (10d)
C = 0.3): À15.8°.
Phenol 8 (150 mg, 0.72 mmol) in DMF (4 mL) was combined with
4-fluoro-2-butynyl methanesulfonate (145 mg, 0.87 mmol) and
potassium carbonate (300 mg, 2.17 mmol) at 90 °C for 3 h according General
Methods. The residue was purified by flash column chromatography (SiO2:
(R)-4-(3-(cyclopentyloxy)-4-(2-fluoroethoxy)phenyl)pyrrolidin-2-one
(11a)—
2-Fluoroethyl 4-methylbenzenesulfonate (257 mg, 1.18 mmol) and
dichloromethane/methanol 9/1) and recrystallized from ethyl acetate to potassium carbonate (163 mg, 1.18 mmol) was added to a solution of
obtain pyrrolidinone 10d (100 mg, 50%) as a white solid: 1H-NMR
(500 MHz, CDCl3): δH 6.94 (m, 1H), 6.89 (m, 2H), 5.57 (bs, 1H), 5.00 (dt, 2H,
J = 48.5 Hz and 1.6 Hz), 4.87 (dt, 2H, J = 7.4 Hz and 1.6 Hz), 3.89 (s, 3H), 3.79
phenol 9 (140 mg, 0.536 mmol) in DMF (5 mL). The solution was heated
at 90 °C overnight. The reaction mixture was quenched with 10 mL of
water, extracted with AcOEt (3 × 10 mL). The organic layers were
(t, 1H, J = 9.2 Hz), 3.68 (quintet, 1H, J = 8.3 Hz), 3.41 (m, 1H, J = 9.2 Hz and combined and washed with water (2 × 10 mL), dried (Na2SO4) and
J = 7.3 Hz), 2.75 (dd, 1H, J = 16.8 Hz and 8.9 Hz), 2.47 (dd, 1H, J = 16.9 Hz and
8.7 Hz). 13C-NMR (125 MHz, CDCl3): δC = 177.96 (C), 148.71 (C), 146.70 (C),
134.68 (C), 120.44 (d, CH, J = 11.6 Hz), 113.45 (CH), 111.92 (CH), 84.82 (d, C,
J = 11.8 Hz), 81.62 (d, C, J = 22.0 Hz), 70.44 (d, CH2, J = 164.9 Hz), 56.91 (d,
CH2, J = 2.8 Hz), 55.90 (CH3), 49.69 (CH2), 39.72 (CH), 38.17 (CH2). HRMS ES
(+): m/z calcd for C15H17 FNO3 (M + H)+: 278.1192, found: 278.1194. Elemental
Analysis: calcd: C: 64.97; H: 5.82, N: 5.05; found: C: 64.76; H: 5.84; N: 5.22. αd20
(CHCl3, C = 0.27): À17.0°.
evaporated. The residue was purified by flash column chromatography
on silica gel (CH2Cl2 then CH2Cl2/MeOH 85:15) to obtain 11a (85 mg,
52%) as a colorless solid: 1H NMR (500 MHz, CDCl3): δ 6.82 (d, 1H,
J = 8.1 Hz), 6.71 (d, 1H, J = 1.9 Hz), 6.69 (d, 1H, J = 8.1 Hz and J = 1.9 Hz),
6.44 (bs, 1H), 4.71 (m, 1H), 4.65 (ddd, 2H, J = 39.1 Hz, J = 4.3 Hz and
J = 1.8 Hz), 4.15 (ddd, 2H, J = 28.1 Hz, J = 4.3 Hz and J = 1.8 Hz), 3.69 (t, 1H,
J = 8.5 Hz), 3.55 (quintet, 1H, J = 8.3 Hz), 3.31 (dd, 1H, J = 9.3 Hz,
J = 7.5 Hz), 2.64 (dd, 1H, J = 16.9 Hz, J = 9.0 Hz), 2.39 (dd, 1H, J = 16.9 Hz,
www.jlcr.org
Copyright © 2016 John Wiley & Sons, Ltd.
J. Label Compd. Radiopharm 2016, 59 205–213

D. Thomae et al.
J = 9.0 Hz), 1.71–1.82 (m, 6H),1.54 (m, 2H). 13C NMR (125 MHz, CDCl3):
178.13 (C), 149.38 (C), 148.51 (C), 136.81 (C), 119.53 (CH), 117.22 (CH),
115.31 (CH), 82.63 (d, CH2, J = 169.4 Hz), 81.42 (CH), 69.76 (d, CH2,
J = 20.7 Hz), 50.12 (CH2), 40.40 (CH), 38.52 (CH2), 33.23 (CH2), 24.32 (CH2).
HRMS ES(+): m/z calcd for C17H23FNO3 (M + H)+: 308.1662, found:
308.1667. Elemental Analysis: calcd: C: 66.43; H: 7.21; N: 4.56; found: C:
66.36; H: 7.13; N: 4.69. α2d0 (CHCl3, C = 0.25): À28.1°.
(R)-4-(3-(cyclopentyloxy)-4-((4-fluorobut-2-yn-1-yl)oxy)phenyl)
pyrrolidin-2-one (11d)—
At 0 °C, sodium hydride (60% dispersion in oil, 33 mg, 0.84 mmol) was
added portionwise to a solution of phenol 9 (200 mg, 0.76 mmol) in
3 mL of DMF. The reaction mixture was stirred for 20 min at 0 °C and a
solution of 4-Fluoro-2-butynyl methanesulfonate (151 mg, 0.91 mmol) in
DMF (1 mL) was added. The solution was allowed to RT and stirred for
3 h. The reaction mixture was quenched with 10 mL of water, extracted
with AcOEt (3 × 5 mL). The organic layers were combined and washed
with water (2 × 5 mL), dried (Na2SO4) and evaporated. The residue was
purified by flash column chromatography on silica gel (AcOEt then
AcOEt/MeOH 95:5) to obtain 11d (145 mg, 58%) as a colorless solid: 1H
NMR (500 MHz, CDCl3): δ 7.28 (m, 1H), 7.00 (m, 1H), 6.80 (m, 1H), 5.74
(bs, 1H), 4.95–5.07 (m, 3H), 4.81 (m, 2H), 3.78 (m, 1H), 3.67 (m, 1H), 3.41
(m, 1H), 2.75 (m, 1H), 2.50 (m, 1H), 1.85–1.92 (m, 6H),1.62 (m, 2H). 13C
NMR (125 MHz, CDCl3): 178.42 (C), 149.26 (C), 147.13 (C), 137.12 (C),
119.12 (CH), 117.04 (CH), 114.52 (CH), 85.76 (d, C, J = 11.6 Hz), 81.52 (d,
C, J = 22.0 Hz), 81.05 (CH), 71.60 (CH2), 70.94 (d, CH2, J = 165 Hz), 57.84
(CH2), 50.20 (CH2), 40.37 (CH), 38.67 (CH2), 33.21 (CH2), 24.38 (CH2). HRMS
ES(+): m/z calcd for C20H22FNO3 (M + H)+: 332.1662, found: 332.1656.
Elemental Analysis: calcd: C: 68.86; H: 6.69; N: 4.23; found: C: 68.83; H:
6.58; N: 4.20. α2d0 (CHCl3, C = 0.25): À30°.
(R)-4-(3-(cyclopentyloxy)-4-(3-fluoropropoxy)phenyl)pyrrolidin-2-
one (11b)—
At 0 °C, sodium hydride (60% dispersion in oil, 67 mg, 1.68 mmol) was
added portionwise to a solution of phenol 9 (400 mg, 1.53 mol) in 5 mL
of DMF. The reaction mixture was stirred for 20 min at 0 °C and was
added at once to a solution of (1.04 g, 3.0 mmol) in DMF (8 mL). The
solution was allowed to RT and stirred for 3 h. The reaction mixture
was quenched with 10 mL of water, extracted with AcOEt (3 × 10 mL).
The organic layers were combined and washed with water (2 × 5 mL),
dried (Na2SO4) and evaporated. The residue was purified by flash
column chromatography on silica gel (CH2Cl2/Hex 8:2 then CH2Cl2/
MeOH 95:5) to obtain 3-[2-(cyclopentyloxy)-4-(5-oxo-3-pyrrolidinyl)
phenoxy]propyl 4-methylbenzenesulfonate (530 mg, 73%) as
a
colorless solid. The tosyl intermediate (350 mg, 074 mmol) was
dissolved in THF (7 mL), TBAF (1 M THF, 3.9 mL, 3.9 mmol) was added
and the reaction mixture was refluxed for 2 h. The solvent was removed
by evaporation under vacuum. The residue was dissolved in AcOEt
(30 mL) and washed with HCl 2 M (10 mL). The organic phase was dried
(Na2SO4) and evaporated. The residue was purified by flash column
chromatography on silica gel (CH2Cl2/MeOH 95:5) to obtain 11b
(180 mg, 76%) as a colorless solid: 1H NMR (500 MHz, CDCl3): δ 7.09
(bs, 1H), 6.80 (d, 1H, J = 8.0 Hz), 6.69–6.72 (m, 2H), 4.70 (m, 1H), 4.61
(dt, 2H, J = 47.1 Hz and 5.9 Hz), 4.03 (t, 2H, J = 6.1 Hz), 3.69 (dd, 1H,
J = 9.3 Hz, J = 8.6 Hz), 3.55 (quintet, 1H, J = 8.0 Hz), 3.33 (dd, 1H,
J = 9.6 Hz, J = 7.5 Hz), 2.65 (dd, 1H, J = 16.9 Hz, J = 9.0 Hz), 2.41 (dd, 1H,
J = 16.9 Hz, J = 9.0 Hz), 2.10 (dquintet, 2H, J = 25.8 Hz, J = 6.0 Hz), 1.73–1.84
(m, 6H), 1.54–1.59 (m, 2H). 13C NMR (125 MHz, CDCl3): 178.05 (C), 148.51
(C), 148.41 (C), 135.55 (C), 119.07 (CH), 115.26 (CH), 114.71 (CH), 80.85
(CH), 80.82 (d, CH2, J = 163.1 Hz), 65.28 (d, CH2, J = 21.2 Hz), 49.81 (CH2),
39.88 (CH), 38.26 (CH2), 32.76 (CH2), 30.53 (d, CH2, J = 19.9 Hz), 23.85 (CH2).
(R)-4-(3-(cyclopentyloxy)-4-(2-(2-fluoroethoxy)ethoxy)phenyl)
pyrrolidin-2-one (11e)—
At 0 °C, sodium hydride (60% dispersion in oil, 30 mg, 0.76 mmol) was
added portionwise to a solution of phenol 9 (180 mg, 0.69 mmol) in
4 mL of DMF. The reaction mixture was stirred for 20 min at 0 °C and a
solution of 2-(2-fluoroethoxy)ethyl 4-methylbenzenesulfonate (277 mg,
1.06 mmol) in DMF (1 mL) was added. The solution was allowed to RT
and stirred for 4 h. The reaction mixture was quenched with 10 mL of
water, extracted with AcOEt (3 × 10 mL). The organic layers were
combined and washed with water (2 × 5 mL), dried (Na2SO4) and
evaporated. The residue was purified by flash column chromatography
on silica gel (CH2Cl2/MeOH 95:5) and recrystallized in AcOEt/Hexane to
obtain 11e (130 mg, 54%) as a colorless solid: 1H NMR (500 MHz, CDCl3):
δ 6.84 (d, 1H, J = 8.0 Hz), 6.73 (m, 2H), 4.76 (m, 1H), 4.55 (dt, 2H, J = 47.7 Hz
and 4.1 Hz), 4.50 (m, 1H), 4.11 (m, 2H), 3.85 (m, 3H), 3.79 (m, 1H), 3.72 (m,
1H), 3.58 (m, 1H), 3.35 (m, 1H), 2.64–2.70 (m, 1H), 2.41–2.46 (m, 1H), 1.77–
1.85 (m, 6H), 1.59 (m, 2H). 13C NMR (125 MHz, CDCl3): 174.48 (C), 149.93
(C), 148.76 (C), 136.17 (C), 119.47 (CH), 115.98 (CH), 115.10 (CH), 83.55
(d, CH2, J = 169 Hz), 81.23 (CH), 71.02 (d, CH2, J = 20 Hz), 70.35 (CH2),
69.84 (CH2), 50.25 (CH2), 40.30 (CH), 38.72 (CH2), 33.18 (CH2), 24.28
(CH2). HRMS ES(+): m/z calcd for C19H27 F NO4 (M + H)+: 352.1924, found:
352.1933. Elemental Analysis: calcd: C: 64.94; H: 7.46; N: 3.99; found: C:
64.89; H: 7.46; N: 4.08. α2d0 (CHCl3, C = 0.18): À22.9°.
HRMS ES(+): m/z calcd for
C
18H25FNO3 (M + H)+: 322.1818, found:
322.1813. Elemental Analysis: calcd: C: 67.27; H: 7.53; N: 4.36; found:
C: 67.52; H: 7.58; N: 4.44. α2d0 (CHCl3, C = 1.38): À21.5°.
(R,E)-4-(3-(cyclopentyloxy)-4-((4-fluorobut-2-en-1-yl)oxy)phenyl)
pyrrolidin-2-one (11c)—
At 0 °C, sodium hydride (60% dispersion in oil, 33 mg, 0.84 mmol) was
added portionwise to a solution of phenol 9 (200 mg, 0.76 mmol) in
3 mL of DMF. The reaction mixture was stirred for 20 min at 0 °C and
(R)-4-(3-(cyclopentyloxy)-4-(fluoromethoxy)phenyl)pyrrolidin-2-one
(11f)
was added at once to
a
solution of (2E)-4-fluoro-2-butenyl
4-methylbenzenesulfonate (222 mg, 0.91 mmol) in DMF (3 mL). The
To a cooled (0 °C) solution of phenol 8 (200 mg, 0.97 mmol) in DMF (5 mL)
solution was allowed to RT and stirred for 2 h. The reaction mixture was was added sodium hydride (60% dispersion in oil, 43 mg, 1.06 mmol).
quenched with 10 mL of water, extracted with AcOEt (3 × 5 mL). The
organic layers were combined and washed with water (2 × 5 mL), dried
After stirring for 20 min the mixture was added to a solution of
fluoromethyl 4-methylbenzenesulfonate (216 mg, 1.06 mmol) in DMF
(Na2SO4) and evaporated. The residue was purified by flash column (1 mL). The mixture was allowed to warm to room temperature over 4 h
chromatography on silica gel (CH2Cl then CH2Cl2/MeOH 95:5) and and then heated to 90 °C for 18 h. After cooling to room temperature
recrystallized in AcOEt/Hexane to 11c (130 mg, 51%) as a colorless solid: ware (10 mL) was added and the mixture was extracted with ethyl
1H NMR (500 MHz, CDCl3): δ 6.85 (d, 1H, J = 8.2 Hz), 6.77 (m, 2H), 6.60 (bs,
1H), 6.05 (m, 2H), 4.96 (m, 1H), 4.87 (m, 1H), 4.79 (m, 1H), 4.59 (m, 2H), 3.77
acetate (3 × 10 mL). The combined organic layers were washed with
water (2 × 5 mL), dried (Na2SO4) and evaporated under reduced pressure.
(m, 1H), 3.77 (m, 1H), 3.40 (m, 1H), 2.72 (dd, 1H, J = 16.9 Hz, J = 8.9 Hz), The aqueous phase was extracted with AcOEt (2 × 50 mL). The organic
2.48 (dd, 1H, J = 16.9 Hz, J = 8.9 Hz), 1.83–1.91 (m, 6H),1.64 (m, 2H). 13C
layers were combined, dried (Na2SO4) and evaporated. The residue was
NMR (125 MHz, CDCl3): 178.18 (C), 149.00 (C), 148.48 (C), 136.05 (C),
purified by flash column chromatography on silica gel (AcOEt/Hex 8:2
130.32 (d, CH, J = 11.8 Hz), 127.48 (d, CH, J = 17.08 Hz), 119.34 (CH), then AcOEt/MeOH 95:5) to obtain 11f (47 mg, 15%) as a colorless solid:
115.83 (CH), 114. 94 (CH), 83.03 (d, CH2, J = 162.7 Hz), 81.22 (CH), 69.38
1H NMR (500 MHz, CDCl3): δ 7.19 (bs, 1H), 7.08 (d, 1H, J = 8.1 Hz), 6.81
(CH2), 50.14 (CH2), 40.39 (CH), 38.54 (CH2), 33.24 (CH2), 24.36 (CH2). HRMS (d, 1H, J = 1.7 Hz), 6.78 (dd, 1H, J = 8.1 Hz and 1.7 Hz), 5.65 (d, 2H,
ES(+): m/z calcd for C19H25FNO3 (M + H)+: 334.1818, found: 334.1830.
Elemental Analysis: calcd: C: 68.45; H: 7.26; N: 4.20; found: C: 68.20; H:
7.15; N: 4.14. α2d0 (CHCl3, C = 0.5): À28°.
J = 54.8 Hz), 4.79 (m, 1H), 3.77 (m, 1H), 3.63 (m, 1H), 3.39 (m, 1H), 2.73
(m, 1H), 2.48 (m, 1H), 1.79–1.90 (m, 6H), 1.63 (m, 2H). 13C NMR
(125 MHz, CDCl3): 178.35 (C), 149.22 (C), 145.89 (C), 139.06 (C), 119.76
J. Label Compd. Radiopharm 2016, 59 205–213
Copyright © 2016 John Wiley & Sons, Ltd.
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D. Thomae et al.
(CH), 119.22 (CH), 114.41 (CH), 102.31 (d, CH2, J = 217.3 Hz), 81.01 (CH),
50.16 (CH2), 40.41 (CH), 38.65 (CH2), 33.20 (CH2), 24.34 (CH2). HRMS ES
(+): m/z calcd for C16H21FNO3 (M + H)+: 294.1505, found: 294.1502.
Elemental Analysis: calcd: C: 65.61; H: 6.87; N: 4.78; found: C: 65.39; H:
6.96; N: 4.65. α2d0 (MeOH, C = 0.5): À12°.
(R)-4-(3-(cyclopentyloxy)-4-methoxyphenyl)-1-(2-fluoroethyl)pyrroli-
din-2-one (16)—
At 0 °C, sodium hydride (60% dispersion in oil, 16 mg, 0.4 mmol) was
added portionwise to a solution of R-(À)-rolipram 1 (0.1 g, 0.36 mmol) in
2 mL of DMF. The reaction mixture was stirred for 45 min at 0 °C and a
solution of 2-fluoroethyl 4-methylbenzenesulfonate (79 mg, 0.36 mmol) in
DMF (1 mL) was added dropwise. The solution was stirred at RT for 3 h. To
complete the reaction, 8 mg of sodium hydride was added and the solution
was stirred at RT overnight. The reaction mixture was quenched with
10 mL of water, extracted with AcOEt (3 × 20 mL). The organic layers
were combined and washed with water (2 × 5 mL), dried (Na2SO4)
and evaporated. The residue was purified by flash column
chromatography on silica gel (CH2Cl2/MeOH 95:5) to obtain 16
(60 mg, 52%) as a colorless oil: 1H NMR (500 MHz, CDCl3): δ 6.74 (d,
1H, J = 7.9 Hz), 6.67 (m, 1H), 4.69 (m, 1H), 4.65 (heptuplet, 1H,
J = 3.2 Hz),4.53 (dddd, 2H, J = 47.4 Hz, J = 5.7 Hz, J = 4 Hz, J = 2 Hz), 3.78
(m, 1H), 3.74 (s, 3H), 3.59 (m, 1H), 3.54 (m, 1H), 3.43 (m, 1H), 2.73 (dd,
1H, J = 16.9 Hz and 8.9 Hz), 2.46 (dd, 1H, J = 16.9 Hz and 8.9 Hz), 1.73–
1.85 (m, 6H), 1.51–1.54 (m, 2H). 13C NMR (125 MHz, CDCl3): 174.68 (C),
149.53 (C), 148.32 (C), 135.13 (C), 119.12 (CH), 114.05 (CH), 112.61
(CH), 83.00 (d, CH2, J = 168 Hz), 80.94 (CH), 56.53 (CH3), 56.34 (CH2),
49.98 (CH2), 43.61 (d, CH2, J = 19.6 Hz), 39.28 (CH), 37.55 (CH2), 33.20
(CH2), 24.42 (CH2). HRMS ES(+): m/z calcd for C18H25NO3F (M + H)+:
322.1818, found: 322.1819. Elemental Analysis: calcd: C: 67.27; H: 7.51;
N: 4.36.; found: C: 66.66; H: 7.51; N: 4.44. α2d0 (MeOH, C = 0.1): À6.7°.
(R,E)-4-(3-(cyclopentyloxy)-4-((3-iodoallyl)oxy)phenyl)pyrrolidin-2-
one (11g)—
At 0 °C, sodium hydride (60% dispersion in oil, 47 mg, 1.18 mmol) was
added portionwise to a solution of phenol 9 (280 mg, 1.08 mmol) in
6 mL of DMF. The reaction mixture was stirred for 20 min at 0 °C and a
solution of 2-(E)-3-(tributylstannyl)-2-propenyl 4-methylbenzene
sulfonate (151 mg, 0.91 mmol) in DMF (3 mL) was added. The solution
was allowed to RT and stirred for 1 h30. The reaction mixture was
quenched with 10 mL of water, extracted with AcOEt (3 × 15 mL). The
organic layers were combined and washed with water (2 × 5 mL), dried
(Na2SO4) and evaporated. The residue was purified by flash column
chromatography on silica gel (CH2Cl2 then CH2Cl2/MeOH 95:5) to obtain
(R,E)-4-(3-(cyclopentyloxy)-4-((3-(tributylstannyl)allyl)oxy)phenyl)pyrrolidin-
2-one (570 mg, 58%) as a colorless oil. This intermediate (400 mg,
0.678 mmol) was dissolved in CH2Cl2 (3 mL), iodine (63 mg, 0,248 mmol)
was added and the solution was stirred overnight at RT. The reaction
was quenched with a saturated solution of sodium thiosulfate and
extracted with CH2Cl2 (2 × 10 mL). The organic layers were combined,
dried (Na2SO4) and evaporated. The residue was purified by flash
column chromatography on silica gel (CH2Cl2 then CH2Cl2/MeOH
95:5) to obtain 11g (270 mg, 93%) as a colorless solid: 1H NMR
(R)-4-(3-(cyclopentyloxy)-4-([18F]fluoromethoxy)phenyl)pyrrolidin-2-
(500 MHz, CDCl3): δ 6.79 (d, 1H, J = 8.2 Hz), 6.69–6.74 (m, 3H), 6.47 one (d2-[18F]11f)
(dt, 2H, J = 14.6 Hz and 1.4 Hz), 4.73 (quintet, 1H, J = 4.0 Hz), 4.42
d2-[18F]Fluoromethyl tosylate was synthesized as previously described.33
(dd, 2H, J = 5.4 Hz, J = 1.5 Hz), 3.72 (t, 1H, J = 8.8 Hz), 3.58 (quintet,
1H, J = 8.3 Hz), 3.34 (dd, 1H, J = 9.4 Hz, J = 7.5 Hz), 2.67 (dd, 1H,
J = 16.9 Hz, J = 9.0 Hz), 2.43 (dd, 1H, J = 16.9 Hz, J = 9.0 Hz), 1.76–1.87
(m, 6H), 1.57–1.62 (m, 2H). 13C NMR (125 MHz, CDCl3): 177.66 (C),
148.77 (C), 147.60 (C), 141.11 (C), 136.12 (CH), 118.86 (CH), 116.09
(CH), 114.31 (CH), 80.69 (CH), 79.13 (CH), 71.52 (CH2), 49.66 (CH2),
39.97 (CH), 38.06 (CH2), 32.82 (CH2),23.95 (CH2). HRMS ES(+): m/z
calcd for C18H23INO3 (M + H)+: 428.0723, found: 428.0717. Elemental
Analysis: calcd: C: 50.60; H: 5.19; N: 3.28; found: C: 50.42; H: 5.15; N:
3.24. α2d0 (CHCl3, C = .365): À21.6°.
The resulting purified d2-[18F]fluoromethyl tosylate was added to a
solution of phenol 9 (1 mg) in DMSO (1 mL) in the presence of cesium
carbonate (Cs2CO3) and heated to 120 °C for 10 min. After cooling to
40 °C the crude product was diluted with water (2 mL) and HPLC eluent
(2 mL) and injected onto a semi-preparative HPLC column (Agilent
Eclipse XDB 250 × 10 mm), using an acetonitrile and ammonium formate
(0.1 M) mobile phase (60/40 v/v) at 4 mL/min. The product-containing
fraction was collected and diluted with water before being loaded onto
t
a Waters C18 light Sep-Pak® cartridge. After washing the cartridge with
water, the product was eluted with ethanol (1 mL) into normal saline
containing ascorbic acid (0.3 mg/mL, 14 mL), before sterilization by
filtration through a membrane filter (0.22 μm) into a sterile final product
vial. Typical decay-corrected radiochemical yields were in the 25–30%
range, with a production time of 85 min, with a specific activity in the
150–340 GBq/μmol range.
Radiochemical purity of the formulated product was tested by
analytical HPLC using a Waters Novapak C18 4.6 × 250 mm analytical
column, eluting with a mixture of methanol/water/triethylamine (60/40/
0.24, v/v/v) at a flow rate of 1 mL/min. The drug mass was calculated
by measuring the UV response of the carrier peak with a calibration curve
of 11f, prepared on the same day. The identity of the product was tested
by co-injection of product with a 2 μg/mL solution of 11f. GC and LAL
test were performed as described above.
(R)-4-(3-Iodo-4-methoxyphenyl)pyrrolidin-2-one (12)—
To a solution of pyrrolidinone 15 (0.34 g, 1.26 mmol) in degassed DME
(15 mL) was added hexadimethyltin (1.34 g, 3.77 mmol) and palladium
tetrakis(triphenylphosphine) and the mixture was heated to flux for 5 h.
After cooling to room temperature the solvent was evaporated under
reduced pressure and the residue was purified by flash column
chromatography (SiO2, ethyl acetate/hexanes, 8/2 then ethyl
acetate/methanol, 9/1) to obtain the aryl-stannane intermediate
(80 mg, 18%) as a white solid. To a solution of this intermediate in
dichloromethane (3 mL) was added iodine (63 mg, 0.248 mmol) and
the solution was stirred at room temperature for 18 h. After the
addition of saturated sodium thiosulfate solution (10 mL) the mixture
was extracted with dichloromethane (2 × 10 mL). The combined
organic layers were dried (Na2SO4) and evaporated under reduced
pressure. The residue was purified by flash column chromatography
(SiO2: ethyl acetate/methanol 97:3) to obtain the pyrrolidinone 12
(56 mg) as a white solid: 1H NMR (500 MHz, CDCl3): δH 7.59 (d, 1H,
J = 1.5 Hz), 7.13 (dd, 1H, J = 8.4 Hz and 1.5 Hz), 6.71 (d, 1H,
J = 8.4 Hz), 6.26 (bs, 1H), 3.80 (s, 3H), 3.68 (t, 1H, J = 8.7 Hz), 3.54
(quintet, 1H, J = 8.7 Hz), 3.29 (t, 1H, J = 8.7 Hz), 2.64 (dd, 1H,
J = 16.9 Hz and 9.0 Hz), 2.37 (dd, 1H, J = 16.9 Hz and 9.0 Hz). 13C
NMR (125 MHz, CDCl3): δC = 177.74 (C), 157.67 (C), 138.23 (CH),
136.70 (C), 128.18 (CH), 111.46 (CH), 86.74 (C), 56.87 (CH3), 49.93
(CH2), 39.50 (CH), 38.34 (CH2). HRMS ES(+): m/z calcd for C11H13INO2S
(M + H)+: 317.9991, found: 317.9992. Elemental Analysis: calcd: C:
41.86; H: 3.81, N: 4.42; found: C: 41.95; H: 3.81; N: 4.49. α2d0 (CHCl3,
C = 0.25): À13.9°.
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