Development of subnanomolar radiofluorinated (2-pyrrolidin-1-yl)imidazo[1,2-b]pyridazine pan-Trk inhibitors as candidate PET imaging probes

Article abstract of DOI:10.1039/c5md00388a

Dysregulation of tropomyosin receptor kinases (TrkA/B/C) expression and signalling is recognized as a hallmark of numerous neurodegenerative diseases including Parkinson's, Huntington's and Alzheimer's disease. TrkA/B/C is known to drive tumorogensis and

Full text of DOI:10.1039/c5md00388a

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Development of subnanomolar radiofluorinated
(2-pyrrolidin-1-yl)imidazoij1,2-b]pyridazine pan-Trk
inhibitors as candidate PET imaging probes†
Cite this: Med. Chem. Commun.,
2015, 6, 2184
Vadim Bernard-Gauthier, a Justin J. Bailey,a Arturo Aliaga,b Alexey Kostikov,c
*
Pedro Rosa-Neto,b Melinda Wuest,a Garrett M. Brodeur,d Barry J. Bedell,ef
a
a
*
Frank Wuest and Ralf Schirrmacher
Dysregulation of tropomyosin receptor kinases (TrkA/B/C) expression and signalling is recognized as a hall-
mark of numerous neurodegenerative diseases including Parkinson's, Huntington's and Alzheimer's disease.
TrkA/B/C is known to drive tumorogensis and metastatic potential in a wide range of neurogenic and non-
neurogenic human cancers. The development of suitable positron emission tomography (PET) radioligands
would allow an in vivo exploration of this versatile potential therapeutic target. Herein, the rational remod-
eling of the amide moiety of a 6-(2-(3-fluorophenyl)pyrrolidin-1-yl)imidazoij1,2-b]pyridazine-3-amide lead
structure to accommodate efficient fluorine-18 labeling led to the identification of a series of fluorinated
Trk inhibitors with picomolar IC50. The ensuing representative radiolabeled inhibitors [18F]16 ([18F]-
( )-IPMICF6) and [18F]27 ([18F]-( )-IPMICF10) constitute novel lead radioligands with about 2- to 3- orders
of magnitude increased TrkB/C potencies compared to previous lead tracers and display favorable selectiv-
ity profiles and physicochemical parameters for translation into in vivo PET imaging agents.
Received 7th September 2015,
Accepted 24th October 2015
DOI: 10.1039/c5md00388a
www.rsc.org/medchemcomm
differentiation within distinct neuronal populations.1–3 Nerve
growth factor (NGF) specifically interacts with TrkA, while
Introduction
The tropomyosin receptor kinase (Trk) family consists of
three structurally analogous tyrosine kinases with pivotal sig-
nificance in the embryonic development and post-natal main-
tenance of the mammalian nervous system. Upon extracellu-
lar binding of cognate specific neurotrophins and subsequent
kinase domain autophosphorylation of the full length recep-
tors, the Trk/neurotrophin protein interplay activates key sig-
nal transduction pathways including Ras/MAPK, PI3K and
PLCγ which spatiotemporally support growth, survival and
brain derived neurotrophic factor (BDNF) and neurotrophin-3
(NT-3) selectively bind TrkB and TrkC respectively (Kd ≈ 1.7–
2.3 × 10−11 M).4 NT-3 was also shown to activate TrkA and
TrkB, albeit with lower efficiency.4 TrkA receptor expression
in the central nervous system (CNS) is mostly circumscribed
to cholinergic neurons of the basal forebrain (BFCNs) and
dorsal root ganglion (DRG).5–7 TrkA density is reduced in
BFCNs and cholinergic projections in the cerebral cortex at
early Alzheimer's disease (AD) stages underlining the involve-
ment of TrkA in the cognitive decline characteristic of the
early onset of AD.8,9 In the periphery, TrkA activation is
involved in nociception and is a validated target for chronic
pain.10 In contrast to TrkA, TrkB/C have comparatively high
expression levels in the CNS with topographies encompassing
most brain regions including cortex, striatum, thalamus and
cerebellum.11–13 In rodents, the NTRK2 (TrkB) family gene
expression includes the full length TrkB.tk+ (herein referred
to as TrkB) and the truncated splice variants TrkB.T1 and
TrkB.T2 which lack the intracellular kinase domain.14,15 TrkB.
T1 has been characterized and shown to be abundantly
expressed in the CNS.16 Specific biological functions of TrkB.
T1 remain elusive but include negative regulation of TrkB.tk+
signalling and BDNF sequestration and translocation.17
The expression ratio of TrkB.tk+/TrkB.T1 are highly regulated
in the embryonic and early postnatal nervous system
a Department of Oncology, University of Alberta, 11560 University Avenue,
Edmonton, AB, T6G 1Z2, Canada. E-mail: bernardg@ualberta.ca
b Translational Neuroimaging Laboratory, McGill Centre for Studies in Aging,
Douglas Mental Health University Institute, 6875 Boulevard LaSalle, Montreal,
QC, H4H 1R3, Canada
c McConnell Brain Imaging Centre, Montreal Neurological Institute,
McGill University, 3801 University Street, Montreal, QC, H3A 2B4, Canada
d Oncology Research, The Children's Hospital of Philadelphia, 3501 Civic Center
Blvd., Philadelphia, PA, 19104-4302, USA
e Biospective Inc., 6100 Avenue Royalmount, Montreal, QC H4P 2R2, Canada
f Research Institute of the McGill University Health Centre, 1001 boulevard
Decarie, Montreal, QC H4A 3J1, Canada
† Electronic supplementary information (ESI) available: Experimental procedures,
supplementary figures, tables, scheme, crystallographic data, radio-HPLC chro-
matograms, and 1H, 13C NMR spectral data. CCDC 1422710. For ESI and crystal-
lographic data in CIF or other electronic format see DOI: 10.1039/c5md00388a
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development. In adult life, mounting evidences support TrkB
isoform dysregulation and aberrant TrkB/BDNF signalling as
a hallmark of neurodegenerative and psychiatric diseases/
conditions including AD, Parkinson's disease (PD),
Huntington's disease (HD), schizophrenia and traumatic
brain injury (TBI).18–22
Though historically explored in the context of neurology,
Trk involvement in tumorigenesis and aggressiveness of
human cancers is also well recognized.23,24 Indeed, the proto-
typical NTRK1 (TrkA) gene was initially identified from a
colon carcinoma leading to a constitutively activated fused
tropomyosin-TrkA protein.25 Besides rearranged/mutated
NTRK gene products, compelling evidence shows that
overexpression of the intact Trk proto-oncoproteins, espe-
cially TrkB, is associated with aggressive tumor growth and
efficacious small molecules to bind the extracellular Trk pro-
teins described thus far suffered extensive metabolism in vivo
and did not permeate the blood brain barrier (BBB) despite
favorable in vitro profiling. More recently, we reported on
the development of low nanomolar pan-Trk inhibitor
[11C]GW441759 and an analogous fluorinated derivative [18F]4.33
Preclinical imaging in rodents confirmed [11C]GW441759 as
the first brain penetrating Trk PET radiotracer but blocking
studies failed to demonstrate specific brain binding at the
tested dose. Both [11C]GW441759 and [18F]4 also successfully
imaged Trk in rat brain sections and human neuroblastoma
tumor samples expressing TrkB in vitro. In addition, the
radiosynthetic methodology to access the fluorinated
demethoxy derivative of the Trk inhibitor GW2580 ([18F]5),
was described.34 Despite its unique selectivity, [18F]5 only dis-
plays moderate potencies towards Trk (IC50s = 135–663 nM)
and lacks necessary physicochemical properties to be suitable
for brain PET imaging (Fig. 1). Those radioligands are cur-
rently being evaluated for peripheral neuroblastoma tumor
imaging by our group. As far as Trk neuroimaging is
concerned, it would be desirable to concentrate efforts on
compounds exhibiting subnanomolar affinities for Trk in
combination with desired properties for PET ligand develop-
ment (including Bmax/Kd > 10, clog D7.4 in the range of 2.0–
3.5, topological polar surface area (TSPA) < 80 Å2, hydrogen
bond donor (HBD) ≤ 1).35 Amenability to fluorine-18 labeling
(t1/2 = 109.8 min; 97% β+; Emax (β+) = 0.64 MeV) would also be
favorable due to the better nuclear properties, including lon-
ger half-life which allow for longer synthesis time and ship-
ping, as compared to carbon-11.
treatment resistance in
a set of neurogenic and non-
neurogenic neoplasms including neuroblastoma and pancre-
atic cancer.26,27 During the last decade, considerable medici-
nal chemistry effort, mostly focused towards intracellular
binding kinase inhibitors, has led to the identification of
diverse and highly potent selective pan-Trk tyrosine kinase
inhibitors (TKIs) as putative cancer therapeutics.28–31
In order to non-invasively assess and monitor TrkA/B/C
levels in the brain and in cancer, our group has sought to
develop 11C- and 18F-labeled Trk-targeted positron emission
tomography (PET) radiotracers suitable for in vivo imaging.
We have previously described both extracellular and intracel-
lular binding PET radioligands, the first of which being the
TrkB selective 7,8-dihydroxyflavone-based probes [18F]1 and
[11C]2 (Fig. 1).32 These radioligands, based on one of the rare
Fig. 1 Structure of Trk agonistic radioligands, tyrosine kinase inhibitor (TKIs) radioligands and fluorophenyl- and difluorophenyl pyrrolidine-
containing Trk TKIs.
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In recent years, diverse scaffolds including heterocycle
cores such as imidazoij1,2-b]pyridazine, pyridoij3,2-d]pyrimidine
and pyrazoloij1,5-a]pyrimidine related through conserved fluo-
rinated 2-phenylpyrrolidines fragments have emerged as Trk-
selective inhibitor pharmacophores (6–8, Fig. 1).36–43 (R)-2-
Phenylpyrrolidine is the preferred enantiomeric building
block. However in most cases where pure (S)-2-
phenylpyrrolidine containing derivatives were characterized,
nanomolar Trk inhibitions were still achieved.37 Moreover,
N-(2-fluoroethyl)acetamide inhibitor (R)-8 and related com-
pounds showed unprecedented picomolar potencies towards
TrkA.37 Very recently, a derivative of 7 was also shown to
achieve tumor regression in a KM12 rat tumor model.42 We
hypothesized that a scaffold derived from compound 8,
which provides a favorable alignment of the required physi-
cochemical parameters for PET radioligand development and
was shown to tolerate numerous 3-carboxamide structural
alterations, would be valuable in the exploration of new Trk
radioligands bearing purposely designed 18F-radiolabeled
amide substituents. Herein, we present the design, syntheses
and first in vitro evaluation of novel 18F-labeled Trk PET
6-(2-(3-fluorophenyl)pyrrolidin-1-yl)imidazoij1,2-b]pyridazine-3-
amide radioligand candidates.
reports also suggest that 18F-labeled fluorocyclobutyl-
containing PET probes may be metabolically more robust
than their linear equivalents.46 Hence, compounds 15 and 16
along with the linear N-fluoroalkyl amides 13 and 14 were
synthesized in good yield via HATU coupling between 9 and
the corresponding amines (Table 1). In order to assess the
impact of the amide proton towards Trk binding, the
3-azetidine amide 17 was synthesized as the closest cyclized
structural analog to 13. Direct amidation of the ester interme-
diate 10 also efficiently provided the simpler derivatives 11
and 12.
Using docking simulations, congruent binding poses to
TrkA/B/C were initially obtained in both the inactive (DFG-
out) and active (DFG-in) conformations for 16 (R and S; Fig.
S1 and S2; ESI†). Choi et al.42 recently described X-ray
cocrystal structures of (R)-enantiomer derivatives of 7 bound
to TrkA and TrkC in DFG-in and DFG-out conformations and
suggested that the favoured binding mode likely depends
upon the substituent pattern of the imidazoij1,2-b]pyridazine
core. The binding modes of 16 and derivatives thereof
obtained in our in silico experiments corroborate those X-ray
results with regard to the binding with the active Trk kinases.
In this conformation, the N1 from the imidazoij1,2-
b]pyridazine interacts via H-bonding with the backbone nitro-
gen of Met592/636 (TrkA/TrkB) from the hinge and provides
excellent overlap of the fluorophenyl ring to the ribose bind-
ing pocket including π-stacking with the proximal Phe521/
565.47,48 In this model, the amide substituent is pointing
towards the solvent region and rotation of the C3–CIJcarbonyl)
can lead to the amide carbonyl or NH directed at the hinge
(Fig. S1†). The predicted binding pose of the representative
compound 27 (vide infra) is shown in Fig. 2.
With the carbonyl pointing outward, additional water-
mediated H-bonds to residue Met592/636 may occur while a
strong intramolecular H-bond is possible between the amide
proton and N5. It is likely that the binding of this
preorganized rigidified low energy conformation to Trk in the
DFG-in mode explains in part the increased potencies
observed between inhibitors such as 8 versus 7 due to a lower
entropic penalty. This binding conformation is also consis-
tent with the disclosure of highly potent related macro-
cyclized Trk inhibitors.49
In agreement with the plausible binding mode elucidated,
the synthesis of potential inhibitors incorporating larger
amide substituents was undertaken. Compounds bearing a
4,4-difluorocyclohexyl group and a N-2-(2-fluoroethoxy)ethyl
chain were synthesized. The amine 30 required for the syn-
thesis of 19 was obtained in two steps from 2-fluoroethyl
4-methylbenzenesulfonate 28 (Scheme 1A). Fluorobenzyl-
(20–22) and fluorophenylamide (23–27) compounds were also
obtained. The required aniline 34 for the synthesis of 27 was
Results and discussion
A priority of this work was to explore and identify the optimal
fluorinated 3-carboxamide building blocks integrated into the
(2-pyrrolidin-1-yl)imidazoij1,2-b]pyridazine lead structure to
yield Trk inhibitors of highest possible affinity. As another
important premise, the identified compounds should be eas-
ily labeled in one step with [18F]fluoride to facilitate applica-
bility. Since both (R)- and (S)-substituted pyrrolidines were
previously shown to be highly potent and the racemic build-
ing block is readily available, we selected the racemic
2-(3-fluorophenyl)pyrrolidine motif with the core structure of
lead 8 as a starting point in our structure activity relationship
(SAR) study.44 Following procedures previously described,41
this approach allowed the preparation of a sufficient amount
of the key 3-carboxylic acid intermediate 9 for further derivati-
zation (Table 1, see Scheme S1; ESI†).
Amide moiety design for radiolabeling and chemistry
It was noted that despite the excellent potency attained with
the N-(2-fluoroethyl)-amide substituent in 8 as reported in
patent literature, the synthesis of the 18F-labeled counterpart
of this motif would require a multistep approach using
2-[18F]-fluoroethylamine which in turn would ultimately limit
the later use of the resulting radioligand in a clinical set-
ting.45 Initial SAR efforts therefore focussed on introducing
small aliphatic fluorinated chains attainable as 18F-iso-
topologues via simple SN2-type radiofluorination but which
would not undergo intramolecular five-exo-tet cyclisation to
the oxazoline upon prior functionalization with a leaving
group or during the radiolabeling. In this context structurally
analogous fluorocyclobutyl moieties were selected. Previous
prepared via
a protection/DAST fluorination/deprotection
sequence (Scheme 1B). It was anticipated that compounds
20–26 would be accessible for radiolabeling using direct
radiofluorination of non-activated arenes bearing iodonium
ylides leaving groups if required.50,51 Compound 27 was
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Table 1 SAR and in vitro enzymatic activities and physicochemical data for imidazoij1,2-b]pyridazines Trk inhibitors
a
IC50 (nM)
Bmax/IC50
Bmax/IC50
clog TPSA
(Å2)
IC50TrkA/IC50TrkB
;
(caudate putamen/ (whole brain
Cpd
R
P
TrkA
TrkB
TrkC
1.37
IC50TrkA/IC50TrkC cortex; TrkA)b
average; TrkB/C)b
11.0
10
2.39 59.73 17.7
1.66 70.73 >10 000
1.16 76.52 12.9
1.51 62.53 7.66
2.36
7.5; 12.9
0.8/0.1
c
c
c
9
3250
1.31
1880
0.943
0.374
—
—
—
11
12
9.8; 13.7
1.1/0.2
1.8/0.3
19.8
46.0
0.565
13.6; 20.5
13d
1.70 62.53 1.11 0.13 0.037 0.013 0.080 0.030 30.0; 13.8
8.9/1.8
703
14d
15d
2.15 62.53 2.48 0.96 0.105 0.043 0.115 0.034 23.6; 21.6
2.28 62.53 2.69 0.24 0.120 0.021 0.163 0.011 22.4; 16.5
4.0/0.8
3.7/0.7
248
217
16d
17
2.08 62.53 1.91 0.41 0.076 0.035 0.076 0.035 25.1; 25.1
5.2/1.0
0.1/0.01
9.1/1.3
342
1.1
1.93 53.74 140
3.06 62.53 1.50
22.8
13.8
6.1; 10.1
11.4; 9.7
18
0.132
0.154
197
19
20
1.45 71.76 4.31
2.88 62.53 3.53
0.926
1.09
0.440
0.580
4.7; 9.8
3.2; 6.1
3.2/0.5
3.9/0.6
28.1
23.8
21
2.88 62.53 2.76
0.509
0.299
5.4; 9.2
4.9/0.7
51.1
22
23
2.91 71.76 11.2
3.17 62.53 5.00
1.96
0.950
0.408
0.233
5.7; 11.8
8.6; 12.3
10.5; 20.6
1.2/0.2
2.7/0.4
13.3
44.5
0.584
0.456
24
3.1
71.76 4.80
2.8/0.4
4.8/1.0
57.0
199
25d
3.17 62.53 2.06 0.17 0.131 0.047 0.214 0.093 15.7; 9.6
26d
27d
a
3.17 62.53 1.94 0.90 0.080 0.012 0.095 0.013 24.3; 20.4
5.1/1.0
4.7/1.0
325
262
3.61 62.53 2.10 0.34 0.099 0.008 0.127 0.005 21.2; 16.5
b
[γ-33P]ATP-based enzymatic assay performed by Reaction Biology. Calculated with Bmax value for the caudate putamen for TrkA and from
c
d
the whole brain for TrkB from ref. 55 and the IC50s from TrkA and TrkB respectively. Not determined. Values accompanied by standard
deviation were averaged from three independent experiments.
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Scheme 1 Reagents and conditions: (A) (a) N-boc-ethanolamine, NaH,
DMF, rt, 12 h (50%). (b) TFA, CH2Cl2, rt, 1 h, then HCl (1 M in diethyl
ether) (98%). (B) (c) Boc2O, Et3N, THF, 0 °C – rt, 16 h (85%). (d) DAST,
CH2Cl2, −78 °C, 30 min then 0 °C, 6 h (86%). (e) TFA, CH2Cl2, 0 °C – rt,
5 h (93%).
Fig. 2 Predicted binding poses for (R)-27 bound to TrkA/B in DFG-in
conformation. (A) Docking of (R)-27 to the ATP binding site of TrkA
(PDB 4PMT). (B) Surface model of (R)-27 docked to TrkA (PDB 4PMT).
(C) Superposition of TrkB (pale gray, DFG-in, PDB 4AT3) and the
docking of (R)-27 with TrkA (dark gray, PDB 4PMT). The TrkA (blue)
and TrkB (cyan) glycine-rich loops are highlighted.
Fig. 3 Single-crystal X-ray structure of (R)-22; ellipsoids drawn at 30%
probability.
designed to be readily accessible using conventional SN2
radiofluorination. Compounds 22 and 26 also bear alter-
native anisol moieties suitable for carbon-11 labelling
using [11C]CH3I. The suggested low energy intramolecular
H-bonded U-shaped conformation was illustrated by single
crystal X-ray diffraction of the representative compound (R)-
22 (obtained from the racemate; Fig. 3).
inhibitor for all Trk proteins was derivative 13 containing the
2-fluoroethyl amide lateral chain with an IC50 of 1.11 nM for
TrkA, 0.037 nM for TrkB and 0.080 nM for TrkC. To our satis-
faction, replacement of the linear chain of 13 with the fluoro-
cyclobutyl found in 16 did not significantly alter the excellent
potencies observed (1.05 to 2.05-fold reduction). Elongation
of the amide chains in 14 and 15 by one additional carbon
only slightly affected the potencies. Compound 12 displayed
subnanomolar and low nanomolar potencies for TrkB/C and
TrkA respectively while being ~2-fold more potent than the
primary amide 11 for all Trks. While the ester 10 displayed
In vitro binding studies
The inhibitory activity of compounds 9–27 was obtained
using a [γ-33P]ATP-based enzymatic assay.52 The TrkA/B/C
assay data is summarized in Table 1. The most potent
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significantly lower, yet still low nanomolar potencies, the
presence of a carboxylate in 9 was highly detrimental to the
affinity. As predicted above, the removal of the amide proton
in 17 had a negative effect despite this inhibitor being the
closest analog of 13 with respect to all other structural ele-
ments (616-fold decreased potency for TrkB). As expected,
good tolerance of the sterically demanding gem-
difluorocyclohexane (compound 18), extended linear chain
(compound 19) and aryl-containing derivatives (compounds
20–27) was also observed. The best potencies in the aryl-
containing derivatives were attained by fluorophenyl – com-
pared to fluorobenzylamides. Inhibitors 13 and 16 were
found to match the Trk activity of staurosporine – to our
knowledge, the most potent Trk inhibitor currently available
(Fig. S20†). All compounds also show favorable clog P and
TPSA values for translation into PET tracers (Table 1).
Overall, we identified a total of 8 inhibitors (13–16, 18,
25–27) displaying approximately 200 pM potencies or lower
for TrkB/C and low nanomolar potencies for TrkA with
radiotracer-favorable properties. These most potent deriva-
tives, except for 18, were also found to display the best selec-
tivity profile in the assayed conditions (TrkB/C versus TrkA,
Fig. S21†).53 Due to the extensive sequence conservation in
the 40 residues likely to be involved in binding at the ATP
site (DFG-in) between TrkA and TrkB (95%) and TrkB and
TrkC (100%), it is largely accepted that Trk isoform selectivity
for ATP competitive inhibitors will be challenging to
attain.28,48 The observed moderate selectivity here may be
related to the organization of the glycine-rich loops (Fig. 2C)
or the difference in conformational restriction of the hinge in
TrkA versus TrkB as previously suggested.48 While the
development of a pan-Trk PET radioligand would be highly
valuable, selectively imaging TrkB/C independently of TrkA
and vice versa would be even more desirable. In imaging
studies, the specific PET signal is contingent on both the
receptor density (Bmax) and affinity of the tracer for the spe-
cific target.54 Hence, for Trk neuroimaging, visualizing TrkB/
C selectively will likely be facilitated by the drastic differences
in Bmax and topographies of Trks in the brain. Binding stud-
ies with [125I]-neurotrophins along with in situ hybridization
experiments have shown that TrkB/C densities largely exceed
that of TrkA.7,13,55 While TrkB/C are largely co-expressed in
all brain regions with an average Bmax > 26 nM, TrkA expres-
sion is mostly restricted to the striatum with a Bmax < 9 nM.
Therefore, the (TrkB/C)/TrkA Bmax ratios for all brain regions
range from 3 in the striatum to ≫10 in most other brain
regions. It should then be expected that, even in the absence
of selectivity, the binding of a PET radiolabeled Trk inhibitor
should mostly reflect the TrkB/C distribution. This implies
that selective visualization of TrkA in the brain would require
a highly selective probe to overcome those Bmax differences.
With our most potent and selective compounds (13–16, 18,
25–27), using IC50 values as a substitute for Kd, this translates
into substantial whole brain average binding potentials for
TrkB/C and significantly lower values for TrkA (striatum;
Table 1).
Compounds 16 and 27 were selected for initial evaluation
based on their potencies and expected labeling in one step.
The selectivity profile of the two inhibitors was first
performed in the presence of 0.1 μM of compounds on a
panel of 20 kinases (Table 2). This initial off-Trk kinase cov-
erage included targets from different kinase groups with an
Table 2 Kinase profiling of 16 and 27
Kinase target
Inhibitory activity of 16a (% remaining at 0.1 μM SD)
Inhibitory activity of 27a (% remaining at 0.1 μM SD)
CSF-1R
VEGRR-2
ITK
FLT3
ERK1/MAPK3
ABL1
102.53 0.91
110.00 0.54
93.38 0.22
85.07 0.11
103.41 0.08
95.64 0.46
97.01 0.52
66.65 2.02
94.81 1.41
98.46 1.58
95.20 0.92
49.61 0.55
99.44 0.08
85.47 0.13
99.81 1.14
90.67 0.42
105.04 0.28
90.21 1.10
100.30 1.48
104.76 0.57
2.95 0.46b
0.44 0.29b
0.39 0.52b
104.90 3.63
95.05 0.79
97.63 0.80
95.09 1.39
101.28 0.40
91.34 0.64
103.11 0.40
93.12 4.80
97.05 1.78
99.54 0.07
89.00 0.57
79.63 0.13
100.24 62
78.90 0.26
99.52 0.05
99.69 1.43
103.93 0.59
105.87 0.06
94.07 1.97
100.8 0.48
3.37 0.65
c-Src
EGFR
c-MET
P38A/MAPK14
ALK
ERBB2/HER2
JNK1
PDHK1
PDGFRa
JAK1
SYK
RET
BRAF
c-KIT
TrkA
TrkB
TrkC
0.56 0.18
0.58 0.40
a
b
[γ-33P]ATP-based enzymatic assay performed by Reaction Biology. Values relative to DMSO controls; duplicate experiments. Derived from
dose–response curves at 1.11 × 10−07 M (triplicates).
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emphasis on kinases expressed in the nervous system and
neoplasms. Only ERBB2/HER2 and EGFR were significantly
affected by co-incubation of 16 with remaining activities of
50% and 67% respectively. Those interactions were less pro-
nounced in the presence of 27. Other minor reductions in
inhibitory activity were observed with 16 towards FLT3 and
PDHK1 (15% reduction) and 27 with PDHK1 (20% reduc-
tion). Overall both compounds were found to be ≥1000-fold
selective for TrkB/C for all tested kinases.
yield from the intermediate 35 which was synthesized
near-quantitatively from the coupling of
9
with
3-aminocyclobutanol·HCl (Scheme 2). Synthesis of the tosylate
41 was obtained in 45% overall yield and 4 steps from
2-(4-aminophenyl)ethanol (37, Scheme 3). Initial 18F-fluoride
incorporation was optimized manually using standard condi-
tions. The radiosynthesis of [18F]16 proceeded efficiently at
120 °C for 10 min in anhydrous dimethylformamide (DMF)
with 18F fluoride as a Kryptofix-222/K+/[18F]F− complex while
the ideal conditions to obtain [18F]27 required a lower tem-
perature due to the apparent degradation of the tosylate pre-
cursor. Following semi-preparative reverse-phase high perfor-
mance liquid chromatography (HPLC), the radiochemical
yields (RCYs) obtained under those optimal reaction condi-
Radiosyntheses and plasma stability
Radiosyntheses of [18F]16 (named [18F]IPMICF6) and [18F]27
(named [18F]IPMICF10) proceeded from the corresponding
tosylate precursors. Precursor 36 was obtained in 70%
tions were 24.8
2.6% (n = 3) and 18.3
3.7% (n = 3)
Scheme 2 Reagents and conditions: (a) 3-aminocyclobutanol hydrochloride, HATU, DIPEA, DMF, rt, 12 h (99%). (b) TsCl, Et3N, CH2Cl2, rt, 48 h
(70%). (c) Kryptofix-222/K+/[18F]F−, DMF, 120 °C, 10 min, 24.8 2.6% RCY (n.d.c.), (n = 3).
Scheme 3 Reagents and conditions: (a) TBDPSCl, imid., DMF, rt, 16 h (80%). (b) 9, HATU, DIPEA, DMF, rt, 12 h (99%). (c) TBAF, THF, rt, 2 h (89%).
(d) TsCl, Et3N, CH2Cl2, rt, 96 h (64%). (e) Kryptofix-222/K+/[18F]F−, MeCN, 100 °C, 20 min, 18.4 3.7% RCY (n.d.c.), (n = 3).
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[18F]27 on coronal rat brain sections confirmed specific bind-
ing to TrkB/C-rich brain regions. The selected representative
probes from this series, [18F]16 ([18F]IPMICF6) and [18F]27
([18F]IPMICF10), constitute novel potential PET tracers with 2
to 3 orders of magnitude potency improvement compared
with our previous leads [11C]GW441759 and [18F]5, while
possessing various other key properties suitable for conver-
sion into PET tracers. While the radioligands developed in
this work were evaluated as isomeric mixtures, further struc-
tural refinement is currently ongoing in order to deliver pure
pyrrolidine motifs as it is to be expected that those different
enantiomers will also display distinct affinities and poten-
tially distinctive in vivo imaging profiles. Comparative in vivo
imaging studies of [18F]16, [18F]27 and other radiotracers
derived from this work will be reported in due course.
Fig. 4 Representative in vitro autoradiograms from coronal sections
of rat brain showing the binding of
experiments with 16 (1 μM) (successive sections between baseline and
blocking; anterior: left, posterior: right). BSt = brain stem; CC = corpus
callosum; Cer = cerebellum; CP = caudate putamen; Ctx = cortex; H =
hippocampus; Th = thalamus.
[
18F]16 and competition
for [18F]16 and [18F]27 respectively (non-decay-corrected
(n.d.c.) based on injected HPLC activity). For in vitro
autoradiogaraphy experiments, the radiofluorination was suc-
cessfully implemented on
a
Scintomics automated
Acknowledgements
radiosynthesis module using similar procedures as manually
developed. Radioligands [18F]16 and [18F]27 were then respec-
tively obtained in 3% and 8% isolated RCYs (n.d.c.), >98.5%
radiochemical purities (RCP), specific activity (SA) of 163–244
GBq μmol−1 at the end of synthesis (EOS) in a total synthesis
time of 75 min or under (see ESI†). Both tracers were shown
to be highly stable in human plasma in vitro at 37 °C with
only minute amounts of 18F-fluoride (<5%) emerging at 60
min post-incubation (Fig. S5 and S8†).
We thank Dr. Esther Schirrmacher for reading the manuscript
and providing useful comments. We are thankful to Angelina
Morales-Izquierdo for the mass spectrometry analysis. We
thank Marilyn Grand'Maison for the valuable help for the
preparation of figures. We are also grateful to Dave
Clendening and Blake Lazurko from the Edmonton PET Cen-
tre and Robert Hopewell, Miriam Kovacevic and Dean Jolly
from the Montreal Neurological Institute for radionuclide pro-
duction. We thank Bob McDonald and Michael Ferguson for
the X-ray crystallographic service. This work was financially
supported by Canada Foundation for Innovation (CFI) project
no. 203639 to R. S.
In vitro autoradiography
In addition, the autoradiograms generated from the incuba-
tion of [18F]16 with coronal rat brain sections conclusively
matched the high affinity binding sites of [125I]BDNF, [125I]
NT3 and [125I]NT4/5 and the topographies of TrkB and TrkC Notes and references
mRNA hybridization (including cortex, striatum, thalamus
and cerebellum; Fig. 4).13 In those assays, moderate but sig-
nificant specific binding was confirmed with co-incubation of
non-labeled 16 (1.0 μM; Δ ~ −30%) and similar blocking
results were also obtained with the incubation of [18F]27 in
the presence of the selective Trk inhibitor GNF-5837 (Fig. S22†).
The analogous blocking obtained with the racemic 16 and
the structurally unrelated oxindole-based DFG-out inhibitor
GNF-5837 is indicative that both the (R)- and (S)-enantiomers
binds Trk receptors in vitro.
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