Radiosynthesis of (R,S)-[18F]GE387: A Potential PET Radiotracer for Imaging Translocator Protein 18 kDa (TSPO) with Low Binding Sensitivity to the Human Gene Polymorphism rs6971

Article abstract of DOI:10.1002/cmdc.201900023

Translocator protein (TSPO) is a biomarker of neuroinflammation, which is a hallmark of many neurodegenerative diseases and has been exploited as a positron emission tomography (PET) target. Carbon-11-labelled PK11195 remains the most applied agent for imaging TSPO, despite its short-lived isotope and low brain permeability. Second-generation radiotracers show variance in affinity amongst subjects (low-, mixed-, and high-affinity binders) caused by the genetic polymorphism (rs6971) of the TSPO gene. To overcome these limitations, a new structural scaffold was explored based on the TSPO pharmacophore, and the analogue with a low-affinity binder/high-affinity binder (LAB/HAB) ratio similar (1.2 vs. 1.3) to that of (R)-[¹¹C]PK11195 was investigated. The synthesis of the reference compound was accomplished in six steps and 9 % overall yield, and the precursor was prepared in eight steps and 8 % overall yield. The chiral separation of the reference and precursor compounds was performed using supercritical fluid chromatography with >95 % ee. The absolute configuration was determined by circular dichroism. Optimisation of reaction conditions for manual radiolabelling revealed acetonitrile as a preferred solvent at 100 °C. Automation of this radiolabelling method provided R and S enantiomers in respective 21.3±16.7 and 25.6±7.1 % decay-corrected yields and molar activities of 55.8±35.6 and 63.5±39.5 GBq μmol^?1 (n=3). Injection of the racemic analogue into a healthy rat confirmed passage through the blood–brain barrier.

Full text of DOI:10.1002/cmdc.201900023

Accepted Article
Title: Radiosynthesis of (R, S)-[18F]GE387: A Potential PET
Radiotracer for Imaging Translocator Protein 18 kDa (TSPO) with
low binding sensitivity to the human gene polymorphism rs6971
Authors: Luxi Qiao, Emily Fisher, Lindsay McMurray, Selena Milicevic
Sephton, Matthew Hird, Nisha Kuzhuppilly-Ramakrishnan,
David J. Williamson, Xiouyun Zhou, Eryn Werry, Michael
Kassiou, Saijinder Luthra, William Trigg, and Franklin I.
Aigbirhio
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.201900023
Link to VoR: http://dx.doi.org/10.1002/cmdc.201900023
A Journal of
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10.1002/cmdc.201900023
ChemMedChem
FULL PAPER
Radiosynthesis of (R, S)-[18F]GE387: A Potential PET Radiotracer
for Imaging Translocator Protein 18 kDa (TSPO) with low binding
sensitivity to the human gene polymorphism rs6971
Luxi Qiao,†[a] Emily Fisher,†[a] Lindsay McMurray,[a] Selena Milicevic Sephton,*[a] Matthew Hird,[a] Nisha
Kuzhuppilly-Ramakrishnan,[a] David J. Williamson,[a] Xiouyun Zhou,[a] Eryn Werry,[b] Michael Kassiou,[b]
Saijinder Luthra,[c] William Trigg,[c] and Franklin I. Aigbirhio*[a]
[a]
Ms. L. Qiao, Dr. E. Fisher, Dr. L. McMurray, Dr. S. M. Sephton, Dr. M. Hird, Dr. N. Kuzhuppilly-Ramakrishnan, Dr. D. J. Williamson, Dr. X. Zhou, and Prof.
F. I. Aigbirhio
Molecular Imaging Chemical Laboratory, Wolfson Brain Imaging Centre,
Department of Clinical Neurosciences
University of Cambridge
Cambridge Biomedical Campus, CB2 0SZ, United Kingdom
E-mail: sms96@cam.ac.uk and fia20@medschl.cam.ac.uk
Dr. E. Werry, and Prof. M. Kassiou
School of Chemistry
The University of Sydney
Building F11, Eastern Avenue, NSW 2006 Australia
Dr. S. Luthra and Dr. W. Trigg
[b]
[c]
GE Healthcare
Amersham HP7 9LL
United Kingdom
† These authors contributed equally
Supporting information for this article is given via a link at the end of the document.
Abstract:
Introduction
Neuroinflammation, an immune response to neuronal insult, is a
core component of many disorders such as stroke, multiple
sclerosis (MS), neurodegenerative disorders and brain
tumours.[1] There is a major need to understand the role of
neuroinflammation in these pathologies and thereby support the
development of therapeutics. One approach for assessing
neuroinflammation in vivo is by using the imaging technique of
positron emission tomography (PET) based on using
radiopharmaceuticals that are selective for the 18-kDa
translocator protein (TSPO), a protein with five trans-membrane
helical domains that is localized primarily in the outer
mitochondrial membrane.[2,3] Neuroinflammation is at least in
part driven by activation of microglia, the brain’s resident
Translocator protein (TSPO) is a biomarker of neuroinflammation
which is a hallmark of many neurodegenerative diseases and has
been exploited as a PET target. Carbon-11 labelled PK11195 is still
to date the most applied imaging agent for imaging TSPO despite its
short-lived isotope and low brain permeability. Second generation
radiotracers showed a variance in affinity amongst subjects (low, mix
and high affinity binders) caused by the genetic polymorphism
(rs6971) of the TSPO gene. In order to overcome those limitations a
new structural scaffold was explored based on TSPO
pharmacophore and the analogue with LAB:HAB ratio similar (1.2 vs.
1.3) to that of [11C]1 was investigated. The synthesis of reference
compound was accomplished in 6 steps and 9% overall yield and
precursor was prepared in 8 steps and 8% overall yield. The chiral
separation of reference and the precursor was performed using
supercritical fluid chromatography with >95% ee. The absolute
configuration was determined by circular dichroism. Optimisation of
reaction conditions for manual radiolabelling revealed acetonitrile as
a preferred solvent at 100 °C. Automation of this radiolabelling
method provided R- and S- enantiomers in 21.3±16.7% and
25.6±7.1% decay corrected yields and molar activities of 55.8±35.6
and 63.5±39.5 GBq/µmol (n=3). Injection of racemic analogue into a
healthy rat confirmed passage through the blood brain barrier.
macrophages, which is accompanied by
a
significant
upregulation of TSPO. Therefore imaging the TSPO expression
is a powerful approach for investigating neuroinflammation. The
most commonly used TSPO PET radiotracer is R-1-(2-
chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline
carboxamide ((R)-[11C]PK11195, [11C]1, Figure 1), which is a
selective antagonist of TSPO that binds with nanomolar affinity
(9.3 nmol).[1,4] By PET imaging (R)-[11C]1 has been used to show
neuroinflammation in vivo in stroke, neurodegeneration,
traumatic brain injury and neoplasia.[5] However, there are a
number of challenges that hinder the wider usage of [11C]1 in
research and clinical settings, such as low brain
permeability,[1,4,6] high non-specific and variable plasma protein
1
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10.1002/cmdc.201900023
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binding, and the short half-life of carbon-11 (t1/2 = 20 min) which
restricts its use to PET centres that have a cyclotron on-site.
of an optimized TSPO PET radiotracer, which will ideally have
the improved pharmacokinetic properties of the second
generation TSPO radiotracer and be independent of the
observed polymorphism.
In 2004, a study by Okubo et al. described a tetracyclic indole
based pharmacophore (4, Figure 2) with high affinity for TSPO.[7]
N
O
O
NEt2
N
Based on this structural scaffold,
a
tricyclic derivative
N
O
N
[18F]GE180 ([18F]5, Figure 2) was developed with good affinity
for TSPO. From radiotracer retention, biodistribution, and in vivo
metabolic profile studies, [18F]5 showed high specific binding,
good brain uptake, high uptake and retention in olfactory bulb
(TSPO-enriched region) and good clearance from striatum
(region of low TSPO expression) in rats.[8] This radiotracer has
N
N
O
11CH3
O11CH3
N
Cl
O
now been evaluated in humans for imaging TSPO expression.[9–
18F
[
11C]1
11C]PK11195
[
11C]2
11C]PBR28
[
18F]3
[
18F]DPA714
12]
[
[
However, while [18F]5 demonstrated less dependence than other
second-generation
tracers
on
the
single
nucleotide
Figure 1. First and Second generation TSPO PET radiotracers.
polymorphism, its affinity to TSPO is still significantly different
amongst the three binding groups (binding affinity ratio
LABs:HABs = 5.1).[13] Therefore, there is still a need to develop
Due to these limitations of [11C]1, multiple new TSPO
radiotracers have been developed and studied, which have
improved properties for application, including several labelled
with the longer lived radioisotope fluorine-18 (t1/2= 109.9 min).[1]
However, PET studies using these second generation
a
TSPO radiotracer with low sensitivity towards this
polymorphism.
Towards this objective informed by structure-activity design
features a novel series of compounds based on the chemical
scaffold[14] of 5 ((S)-GE180) were developed and synthesised. A
key design feature being that while retaining good affinity to
TSPO, variation at the amide position could influence the
LAB/HAB ratio. From this the compound with most appropriate
properties for development as a potential TSPO PET radiotracer
with low binding sensitivity to the human gene polymorphism
rs6971 was identified as 6 (GE387) for which its enantiomers
were shown to have affinities of 1.04 nM (which we later in this
work, outlined in the Results and Discussion, identified to be the
S form) and 22 nM (identified to be the R form)[14] based on a
rat heart TSPO assay.[15] Herein, we report for the compound 6,
determination of relative binding affinities to confirm low
sensitivity to the genetic polymorphism, synthesis of reference
compound 6 and precursors for radiolabeling with fluorine-18,
separation and identification of enantiomers and finally
development of protocols for the automated radiosynthesis of its
radiolabelled analogue [18F]6 as well as the initial in vivo
preclinical PET.
radiotracers,
such
as
N-(2-(methoxy-11C)benzyl)-N-(4-
phenoxypyridin-3-yl)acetamide ([11C]PBR28, [11C]2, Figure 1),
identified a variance in affinity to these compounds between
subjects. This affinity variation was established to be caused by
a genetic polymorphism (rs6971) found in exon 4 of the TSPO
gene, which results in substitution of alanine for a threonine in
position 147 (A147T). Thus three distinct subject groups have
been identified: high affinity binders (HABs; Ala/Ala), low-affinity
binders (LABs; Thr/Thr), and mixed-affinity binders (MABs;
(Ala/Thr).[1]
Et
O
N
OMe
Et
R
R
N
N
N
18F
X1, X2 = H, F,
Cl, Me
O
18F]5
Y
[
X1
Y = S, SO2, CH2
R = alkyl or
cycloalkyl
X2
Results and Discussion
Et
N
O
H
OMe
Bn
Chemistry
TSPO pharmacophore
For the radiolabelling of [18F]6, we proposed an SN2 nucleophilic
substitution of a good leaving group with [18F]fluoride, for which
reason tosylate 25 and mesylate 26 were envisioned as
potential precursors. Reference compound 6 (Scheme 1) and
the respective radiolabelling precursors 25 and 26 (Schemes 2
and 3) were prepared following previously reported synthesis of
reference compound and mesylate precursors of 6.[16]
4
N
18F
[
This work
18F]6
Figure 2. Pharmacophore derived second generation analogues.
2-Fluoroethanol 7 was tosylated in pyridine to form 2-fluoroethyl
tosylate 8. Tosylate
8 was used to alkylate 2-chloro-5-
While past research has yielded many new radiotracers for
imaging of TSPO, to date only [11C]1 binds highly independent of
TSPO gene polymorphism (e.g., it binds to both LAB and HAB
binding sites equally as indicated by the LAB/HAB ratio of 1,
Table 2), thus prompting further research into the development
methoxyaniline 9 to afford 10 (Scheme 1). The methoxyaniline
10 was then deprotonated with KHMDS in THF and reacted with
a bromide intermediate 11 to give enol 12, which subsequently
underwent cyclisation assisted with zinc chloride in refluxing
2
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10.1002/cmdc.201900023
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diethyl ether to afford the tricyclic product 13, isolated in 31%
yield over two steps. Chloro-analogue 13 was dehalogenated
with hydrogen on activated palladium to obtain ester 14, which
was hydrolyzed to acid 15. The crude acid 15 was reacted with
N-ethylbenzyl amine via an acid chloride intermediate 16 formed
with oxalyl chloride to form the reference standard 6 with a yield
of 28% over four steps.
Pd/C under basic conditions (triethylamine) to give product 23. A
second hydrogenation on crude 23 in the absence of
triethylamine was performed to remove the benzyl protecting-
group to give a crude 24 in quantitative yield. Using crude
alcohol 24, we prepared both crude tosylate 25 and crude
mesylate precursor 26 (Scheme 3) in a reaction with tosyl or
mesyl chloride, with 13% and 12% yield over five steps,
respectively. Tosyl analogue 25 was successfully purified by
recrystallisation from EtOAc to give a crystalline solid (33%).
However, the mesyl precursor 26 required purification by column
chromatography, yielding a viscous oil (37%), thus making tosyl
precursor 25 practically easier to purify and handle than the
mesyl precursor 26.
CO2Et
OR
HO
Br
F
7, R = H
OMe
OMe
a
8, R = Ts
11
b
c
NH
NH2
Cl
Cl
CO2Et
HO
Br
O
9
10
F
OBn
OMe
OMe
H
17
11
OMe
CO2Et
OMe
CO2Et
a
b
NH
NH2
HO
d
Cl
Cl
9
18
N
N
OBn
R
F
Cl
F
13, R = Cl
14, R = H
12
OMe
CO2Et
OMe
CO2Et
OBn
e
HO
c
f
N
N
Cl
OBn
Cl
O
N
OMe
C(O)R
OMe
20
19
HN
d
h
N
N
F
F
O
N
OMe
CO2H
OBn
OMe
6
15, R = OH
16, R = Cl
HN
g
e
N
N
Cl
R1
OR2
Scheme 1. Synthesis of reference compound 6. Reagents and conditions: a.
TsCl, Pyridine, 23 °C, 18 h, 26%; b. 8, NaH, DMF, 100 °C, 18 h, 32%; c.
KHMDS, THF, 23 °C, 4 h; d. ZnCl2, Et2O, reflux, 16 h, 31%; e. H2, Pd/C, EtOH,
23 °C, 18 h; f. NaOH, H2O, EtOH, reflux, 18 h; g. (COCl)2, DMF, 23 °C, 2 h; h.
CH2Cl2, 23 °C, 18 h, 28% (over 4 steps).
21
22, R1 = Cl, R2 = Bn
23, R1 = H, R2 = Bn
24, R1 = H, R2 = H
f
g
Scheme 2. Syntheses of radiolabelling precursors 25 and 26. Reagents and
conditions: a. NaBH(OAc)3, CH2Cl2, 23 °C, 18 h, 74%; b. KHMDS, THF, 23 °C,
1.5 h; c. ZnCl2, Et2O, reflux, 5 days, 85% (over 2 steps); d. NaOH, H2O, EtOH,
80 °C, 18 h; e. HOBt, EDCI, 23 °C, 48 h; f. H2, Pd/C, MeOH, Et3N, 23 °C, 18 h;
g. H2, Pd/C, MeOH, 23 °C, 18 h.
Analogously, 2-chloro-5-methoxyaniline hydrochloride 9 reacted
with benzyloxyacetaldehyde 17 under reductive conditions to
form the amine 18 (Scheme 2) in 74% yield. As with the
synthesis of the reference compound 6, the next alkylation step
used the same previously made bromide intermediate 11 with
KHMDS to yield 19. The crude product was similarly cyclized
using zinc chloride in diethyl ether, to give 20 with 85% yield.
Ester 20 was then hydrolysed using sodium hydroxide in ethanol
and H2O to give acid 21. Although the two step amide formation
via the acid chloride intermediate was successfully used in the
synthesis of the reference compound 6, here we found that a
one-step HOBt/EDCI amide coupling was more efficient for the
synthesis, yielding 22. The reaction utilises two activated ester
derivatives as intermediates, regenerating HOBt and giving urea
as a by-product. However, on a larger scale this route was not
so efficient due to formation of homocoupled by-products.
Compound 22 was dehalogenated in the presence of H2 on
O
N
O
N
OMe
OMe
a
b
24
N
N
OTs
OMs
25
26
Scheme 3. Completion of syntheses of radiolabelling precursors 25 and 26.
Reagents and conditions: a. TsCl, DMAP, Pyridine, 23 °C, 18 h, 13% (over 5
steps); b. MsCl, Et3N, CH2Cl2, 23 °C, 18 h, 12% (over 5 steps).
3
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Since it is well known that enantiomers may have different
pharmacokinetic, pharmacodynamic and safety/toxicity profiles,
we also aimed to investigate the difference between the two
enantiomers of [18F]6 by establishing the chiral separation of
While NMR analysis demonstrated the presence of two
diastereomers in all three product mixtures, TLC analysis of the
ester mixtures using a variety of solvent systems showed the
two diastereomers could be separated readily by column flash
chromatography in two cases, that of L-menthol and (R)-1-
phenyl-2-propynyl analogues. Flash chromatography using
diethyl ether and cyclohexane yielded two diasteriomerically-
enriched samples with both esters 29 (in 78% and 40%
diastereomeric excess) and 30 (in 82% and 35% diastereomeric
excess) (Scheme 4, (S, L)-29, (R, L)-29 and (S, R)-30, (R, R)-
30). To demonstrate presence of a single enantiomer in a
mixture, the chiral L-menthol functionality was first removed
under a variety of basic conditions (Table 1). LCMS analysis
showed near-complete transformation with NaOH/H2O in EtOH
(entry 1), which was the hydrolytic condition used in the
synthesis of the reference compound 6 and precursors 25 and
26. Reaction with LiOH in a mixture of THF/H2O (entry 2) gave a
minor by-product.[17]
both reference compound
6 as well as its radiolabelling
precursor (25). An obvious choice for chiral separation was
found in preparation of diastereomeric mixture by application of
a chiral derivatization group. We identified an acid intermediate
21 for further functionalization with several potential
enantiomerically pure alcohols. The alcohols selected were L-
menthol, (R)-(-)-2-butanol and (R)-1-phenyl-2-propyn-1-ol. The
ester 28 with (R)-(-)-2-butanol was formed in 53% yield using
DBU as a base and a mesylate analogue 27 of the alcohol as an
intermediate. However, a higher yielding and a more direct
method of EDCI/DMAP coupling was used to form the esters
with L-menthol and (R)-1-phenyl-2-propyn-1-ol, which afforded
29 and 30 in 54% and 31% yield, respectively (Scheme 4).
Me
Me
Me
OH
O
O
O
OH
H
O
O
H
OH
Me
O
O
O
H
Me
Me
a
b
N
N
N
Cl
Cl
Cl
OBn
OBn
OBn
(S, L + R, L)-29
column chromatography
Me Me
(S + R)-21
(S, R + R, R)-30
OMs
c
column chromatography
27
O
O
H
O
O
O
O
H
O
O
O
H
column
Me
+ (R, L)-29
N
N
N
chromatography
Cl
Cl
Cl
+ (R, R)-30
(S, S)-28 + (R, S)-28
OBn
OBn
OBn
(S, R)-30
(S, L)-29
(S, S + R, S)-28
see Table 2
d
Me
Me
O
OH
H
O
OH
H
O
O
O
O
O
H
e
Me
N
N
N
Cl
Cl
Cl
+ (R)-21
+ (R)-21
OBn
OBn
OBn
(S)-21
(S)-21
(S, L + R, L)-29
Scheme 4. Separation of enantiomers using derivatization method with chiral alcohols. Reagents and conditions: a. EDCI, DMAP, CH2Cl2, 23 °C, 16 h, 54%; b.
EDCI, DMAP, CH2Cl2, 23 °C, 4 h, 31%; d. DBU, Toluene, 80 °C, 4 h; e. EDCI, DMAP, CH2Cl2, 23 °C, 16 h, 54%; f. CuCl, MeOH, 40 °C, 6 h.
Using K2CO3 in MeOH (entry 3) gave an equal amount of the
desired product and an unidentified by-product,[18] while reaction
with trimethyltin hydroxide (entry 4) showed an incomplete
reaction (63% by LC) even when the reaction time was
increased.[19] To assess the enantiomeric purity of the product
formed, diastereomerically enriched acid was re-esterified with
L-menthol using EDCI/DMAP. To our disappointment, H NMR
analysis indicated complete racemization from all investigated
1
4
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35
25
15
5
hydrolytic reaction conditions. This was further confirmed by
chiral HPLC analysis of the acid samples obtained from
hydrolysis of diastereomerically-enriched ester samples (e.g.,
(S)-21, Scheme 4). Poor stability of the acid 21 in a solution
presumably due to the presence of a highly acidic proton is a
likely explanation for such rapid racemization of the stereocenter.
In the case of the ester analogue 30, while we were able to
obtain diastereomerically enriched samples through multiple
rounds of column flash chromatography in diastereomeric
excess ((S, R)-30, (R, R)-30), a CuCl hydrolysis protocol
described in literature failed to give the enantiomerically
enriched product 21 (Scheme 3).[20] Faced with immediate need
to have reference material and precursor separated into
enantiomers, we turned to a commercial partner who performed
enantiomer separation successfully using SFC for both
(S)-5 (GE180)
GE180
S
(
-
S
GE3
)
8
-
7
6 (GE387)
R-GE387
(R)-6 (GE387)
190
200
210
220
230
240
250
260
270
280
290
300
-5
-15
-25
Wavelength (nm)
reference compound
information for details).
6 and tosylate 25 (See Supporting
Figure 3. CD spectral data for (R)-6 and (S)-6 and comparison with known CD
spectrum of (S)-5.
Table 1. Conditions for the removal of chiral functionality.
Assessment of GE387 binding affinity to Ala147Thr TSPO
Time
(h)
Conversion
Temperature
(°C)
Using an assay based on human embryonic kidney cell lines
stably over-expressing human TSPO WT and TSPO A147T[13]
compound 5 was shown to bind to WT TSPO with a higher
affinity than compound 1, but lost affinity at A147T TSPO (Table
2). Compound (S)-6 bound to WT TSPO with a similar affinity to
compound 1, and both compounds 1 and (S)-6 only showed
minor loss in affinity at A147T TSPO (1.2-1.3x reduction; Table
2). It has been shown for this assay that compounds have lower
binding affinities than those commonly reported at the rat TSPO.
This is also shown for (S)-6: 36.3 nM while 1.04 nM for rat
TSPO.[14]
(% by LCMS)
Conditions
Entry
·····································································
18
90
50
95
63
80
23
23
60
NaOH/H2O, EtOH
LiOH, THF/H2O
K2CO3, MeOH
1
2
3
4
4
3
16
Me3Sn(OH), DCE
Absolute stereochemistry of enantiomers
The absolute stereochemistry of enantiomers of 6 was next
determined using the electronic circular dichroism (CD)
spectrometry whereby CD spectra of 6 were compared with CD
spectrum of 5 for which stereochemistry was already established
(Figure 3). The spectrum of known (S)-5 was composed of a
maximum positive Cotton effect at 200 nm and a corresponding
maximum negative Cotton effect at 226 nm. The enantiomer of 6
which similarly exhibited a maximum positive Cotton effect at
204 nm and a corresponding maximum negative Cotton effect at
228 nm was in analogy assigned as S-isomer. The other isomer
showed the mirror image bisignate band in the CD spectrum and
was therefore identified as R-enantiomer.[21]
Table 2. Binding affinities (Ki) for WT and A147T TSPO.
Ki (nM)
at Ala147Thr at WT (Ala/Ala)
LAB HAB
Ki (nM)
Ratio
Derivative
LAB/HAB Sample Size
·······································································
(S)-5
(S)-6
(R)-1
58.3±6.4
47.3±7.0
36.0±6.8
11.3±0.6
36.3±6.9
29.2±7.3
5.1
1.3
1.2
3
3
3
To correlate enantiomers obtained via the SFC separation to CD
data, the chiral HPLC separation was performed on the Whelk-
O1 column to unambiguously identify first eluting peak as S-
enantiomer (See Supporting information for details).
Radiochemistry
In order to establish suitable reaction conditions for
radiolabelling of [18F]5 manual radiochemical reactions were
performed first. Both precursors 25 and 26 were assessed and
the effect of temperature, solvent and time was investigated
(Table 3).
For all reactions using 25 (entries 1-6), a single radiolabelled
product formed which was identified as [18F]6 via co-injection of
reference compound 6. However, when using 26 (entries 7-12),
a second unidentified radiolabelled product was formed during
the reaction. The amount of this radiolabelled side product which
5
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10.1002/cmdc.201900023
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formed immediately, did not change during the course of the
reaction.
Total radiosynthesis time for the preparation for [18F]6 was 60
min from end of bombardment (EOB). Radiochemical purity was
over 99%, and radiolabelling yield was 20.8±4.5% decay
corrected to EOB or 13.6±2.8 % non-decay corrected, and molar
radioactivity 93.2±50.6 GBq/µmol (n=9) at end of synthesis
(EOS).
Furthermore, the same automated protocol was used for the
radiosyntheses of (R)-[18F]6 and (S)-[18F]6 as follows:
21.3±16.7% and 25.6±7.1% decay corrected yields,
radiochemical purity >98% and molar radioactivities 55.8±35.6
and 63.5±39.5 GBq/µmol (n=3) at the EOS, respectively.
At 80 °C, independent of solvent and precursor low conversion
of <20% of [18F]fluoride to [18F]6 was observed: highest being
18% when MeCN was used as a solvent and mesylate 25 as a
precursor. When the temperature was increased to 100°C or
120°C reactions performed in DMF similarly showed poor
conversion (entries 6 and 12) after 20 min. In DMSO, [18F]6
formed with 3% conversion after 20 min (entry 10) when 26 was
used as precursor, whereas tosylate 25 afforded 47%
conversion to [18F]6 under analogous conditions (entry 4). The
highest conversion in case of both precursors 25 and 26 was
observed after 20 min in MeCN: 81 and 39%, respectively
(entries 2 and 8).
In Vivo PET Analysis
With this result in hand (MeCN as a solvent, 100 °C, 20 min),
next tosylate 25 was used for automated radiolabelling on a GE
Healthcare FXFN TRACERlab. [18F]Fluorination was achieved
with cyclotron-produced [18F]fluoride, in MeCN at 100°C for 20
min. Product [18F]6 was purified by semi-preparative reversed-
phase HPLC.
As a proof-of-principle of our novel radiotracer candidate we
next performed PET scans in male Wistar rats to establish that
racemic [18F]6 enters the brain (Figure 4). The time activity curve
indicates distribution of radioactivity in the brain, albeit in a
modest amount as the investigated animals were healthy.
a.
Table 3. Screened reaction conditions for radiolabelling of [18F]6.
b.
SSUUVV
0
0
3.5
2.0
sagittal
transversal
coronal
Figure 4. a) Time activity curve (TAC), n=2 over the acquisition time of 60 min
and b) summed 30-60 min PET image of racemic [18F]6 in a healthy rat.
Conclusions
Here we investigated the synthesis of a novel TSPO radiotracer
[18F]6, which has high binding affinity for TSPO. Critically we
have established compound 6 has low sensitivity to the human
gene polymorphism rs6971. Based on the synthetic sequence of
an analogue [18F]5, we synthesized the racemate of reference
compound 6 and appropriate radiolabelling precursors 25 and
26 for radiolabeling with fluorine-18. We then explored chiral
6
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10.1002/cmdc.201900023
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The reaction was stirred at RT for 18 h. The reaction was then quenched
by careful addition of ice followed by water (5 mL). The reaction mixture
was extracted into EtOAc (10 mL) and washed with water (3×10 mL), 1M
HCl solution (10 mL), 1M aqueous sodium carbonate (10 mL), and
copper sulfate (2×10 mL). The organic layer was washed with brine (10
mL), dried (MgSO4) and concentrated in vacuo to give 8 as an oil (136
mg, 0.62 mmol, 26%). LC-MS: Rf 1.86 (-ESI) m/z 311.5 ([M + 94]+)1. 1H
NMR (300 MHz, CDCl3): δH 7.81 (2H, d, J = 8 Hz, SO2CCHCH), 7.36 (2H,
d, J = 8 Hz, SO2CCHCH), 4.57 (2H, ddd, J = 3 and 50 Hz, OCH2CH2F),
4.26 (2H, ddd, J = 4 and 30 Hz, OCH2CH2F), and 2.45 (3H, s, CCH3).
The compound was in complete agreement with previously published
data.
resolution using the formation of diastereomeric mixtures, but
were met with disappointing results. With the racemic precursors
in hand, we then optimized radiolabeling conditions under
manual reaction conditions and have subsequently established
the automatic radiolabelling conditions to form both racemic and
enantiomerically pure isomers of [18F]6.
The PET scans
performed indicate that racemic [18F]6 enters the brain. Detailed
studies of biological evaluation of each of the enantiomers of
[18F]6 are currently ongoing in our laboratory and will be reported
in due course.
2-Chloro-N-(2-fluoroethyl)-5-methoxyaniline (10). Commercially available
2-chloro-5-methoxyaniline hydrochloride 9 (5.00 g, 26 mmol, 1 eq) was
dissolved in DMF (60 mL) and sodium hydride (60% dispersion in mineral
oil, 2.30 g, 57 mmol, 2.2 eq) was added. The reaction was stirred for 30
minutes at RT under nitrogen. The tosylate 8 (6.00 g, 31 mmol, 1.2 eq) in
DMF was then added dropwise and the reaction was stirred at RT for 2 h.
The reaction was then heated at 100°C for 18 h. The reaction was
allowed to cool and the solvent was removed under reduced pressure.
The residue was dissolved in EtOAc (100 mL) and washed with water
(5×100 mL). The organic layers were combined, dried (MgSO4) and
concentrated in vacuo to give a brown oil. The crude mixture was then
purified by silica flash chromatography eluting with a gradient of 5-30%
EtOAc/petroleum ether (40-60°C) to yield 10 as a yellow oil (1.69 g, 8.32
mmol, 32%). LC-MS: Rf 2.01 (+ESI) m/z 204.3 ([M + H]+). 1H NMR (300
MHz, CDCl3): δH 7.16 (1H, dm, J = 5 Hz, CHC(OCH3)CHCH), 6.25-6.22
(2H, m, CHC(OCH3)CHCH), 4.64 (2H, dt, J = 5 and 47 Hz, NCH2CH2F),
4.65-4.55 (1H, b, NH), 3.77 (3H, s, OCH3), and 3.48 (2H, dq, J = 5 and
26 Hz, NCH2CH2F). The compound was in complete agreement with
internal GE publication.
Experimental Section
General
All reactions requiring anhydrous conditions were conducted in oven-
dried glass apparatus under an atmosphere of inert gas. Reagents were
purchased from Sigma-Aldrich or Alfa Aesar. Reagents were used
without further purification unless otherwise noted. Triethylamine (Et3N)
was distilled over P2O5 and stored over KOH. Reported density values
are for ambient temperature. Purity of compounds was ≥95% as
determined by analytical HPLC method on Thermo Dionex 3000 HPLC
system. Preparative chromatographic separations were performed on
Material Harvest silica gel 60 (35-75 µm) and reactions followed by TLC
analysis using Sigma-Aldrich silica gel 60 plates (2-25 µm) with
fluorescent indicator (254 nm) and visualized with UV or potassium
permanganate.
1H-NMR spectra were recorded in Fourier transform mode using a Bruker
Avance III 300 MHz FT-NMR spectrometer and the data analysed using
TopSpin program. Spectra were obtained from the specified deuterated
solvents in 5 mm diameter tubes. Chemical shift in ppm is quoted relative
to residual solvent signals calibrated as follows: CDCl3 δH (CHCl3) = 7.26
ppm, δC = 77.2 ppm. Multiplicities in the 1H NMR spectra are described
as: s = singlet, d = doublet, t = triplet, q = quartet, quint. = quintet, s =
sextet, h = heptet, m = multiplet, b = broad; coupling constants are
reported in Hz.
Ethyl 3-bromo-2-hydroxycyclohex-1-ene-1-carboxylate (11). Ethyl 2-
oxocyclohexanecarboxylate S1 was dissolved in diethyl ether and cooled
to 0°C under nitrogen. Bromine was added dropwise over 15 min and the
reaction mixture was allowed to warm to RT over 90 min. The mixture
was slowly poured into ice-cold saturated aqueous potassium carbonate
and extracted with ethyl acetate. The combined organic layers were dried
over magnesium sulfate, filtered, concentrated in vacuo and dried on the
vacuum line for 18 h to afford 11 as a yellow oil. LC-MS: Rt 1H NMR (300
MHz, CDCl3): δH 11.92 (1H, s, OH), 4.93 (1H, t, J = 3.4 Hz, BrCHC(OH)),
4.20 (2H, ddd, J = 1, 7 and 14 Hz, C(O)OCH2CH3), 2.53-2.46 (2H, m,
BrCHCH2CH2CH2), 2.12-2.03 (2H, m, BrCHCH2CH2CH2), 1.79-1.67 (2H,
m, BrCHCH2CH2CH2), 1.24 (3H, t, J = 7 Hz, C(O)OCH2CH3) The
compound was in complete agreement with internal GE publication.
LC-MS data was obtained using Waters Acquity-H/Xevo TQD LC-MS.
Acquity UPLC® BEH C18 1.7 um, 2.1x 50 mm. 0.6 mL/min 4 min method,
5-95% MeCN/H2O with 0.1% Formic Acid. By LC. HRMS were obtained
from the EPSRC Mass Spectrometry Service at the University of
Swansea. Ion mass/charge (m/z) ratios are reported as values in atomic
mass units.
Ethyl-3-((2-chloro-5-methoxyphenyl)(2-fluoroethyl)amino)-2-
hydroxycyclohex-1-ene-1-carboxylate (12). A solution of the aniline 10
(2.20 g, 10.8 mmol, 1 eq) in THF (60 mL) was cooled to -40°C.
Potassium bis(trimethylsilyl)amide (0.5 M solution in toluene, 45.4 mL,
4.5 g, 22.7 mmol, 2.1 eq) was added dropwise and the reaction stirred
for 30 min at -40°C. The carboxylate 11 (2.67 g, 10.8 mmol, 1 eq) in THF
(10 mL) was added dropwise at -40°C. The cooling bath was removed
and the reaction was stirred at RT for 4 h. The reaction was quenched
with brine (100 mL) and extracted into EtOAc (2×200 mL), dried (MgSO4)
and concentrated in vacuo to give 12 as a brown oil (3.70 g, 9.97 mmol)
which was used crude in the next step. LC-MS: Rf 2.29 (+ESI) m/z 372.4
([M + H]+). The compound was in complete agreement with internal GE
publication.
The automated radiosynthesis was performed on a GE Healthcare FXFN
TRACERlab. Semi-preparative purification of radiolabelled material was
performed on a Merck-Hitachi L6200A system equipped with Knauer
variable wavelength detector and an Eberline radiation detector using a
reverse phase column (ACE 160433, C18, 5 µm, 100x10 mm) and
eluting with 48% aq. MeCN at flow rate 5 mL/min. Analytical HPLC
samples were analysed by Agilent HPLC 1100 system equipped with UV
multi-wavelength detector and Raytest Gabi star radiation detector using
reverse phase column (Phenomenex Luna 5u C18 (2) 100A. 250 x 4.6
mm. 675295-11) and eluting with 0-3 min isocratic at 10% aq. MeCN, 3-5
min gradient 10-80% aq. MeCN, 6-12 isocratic 80% aq. MeCN at flow
rate 1.5 mL/min.
Ethyl
8-chloro-9-(2-fluoroethyl)-5-methoxy-2,3,4,9-tetrahydro-1H-
Chemistry
carbazole-4-carboxylate (13). The intermediate 12 (3.71 g, 10.0 mmol, 1
eq) was dissolved in Et2O (100 mL) and zinc chloride (5.45 g, 40.0 mmol,
2-Fluoroethyl 4-methylbenzenesulfonate (8). Commercially available 2-
fluoroethanol 7 (0.141 mL, 153 mg, 2.40 mmol, 1 eq, d = 1.09 g/cm3) was
dissolved in pyridine (2.5 mL) under nitrogen. The solution was stirred at
0°C and tosyl chloride (1.00 g, 5.25 mmol, 2.2 eq) added portionwise to
the solution over a period of 30 min, keeping the temperature below 5°C.
7
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10.1002/cmdc.201900023
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FULL PAPER
4 eq) was added. The reaction was heated at reflux for 16 h. EtOAc (300
mL) was added to dissolve everything and was washed with 2M HCl (200
mL), water (200 mL), 10% aqueous potassium carbonate (200 mL), dried
(MgSO4) and concentrated in vacuo. The crude material was purified by
CH2CH2F, 4-CH), 4.31 (2H, dt, J = 5 and 24 Hz NCH2CH2F), 3.62 (3H, s,
OCH3), 3.83-3.41 (4H, m, N(CH2CH3)2), 2.88-2.61 (2H, m, 1-CH2), 2.20-
1.80 (4H, m, 2- and 3-CH2), and 1.32 (3H, t, J = 7 Hz, N(CH2CH3)2).
silica gel chromatography eluting with
a gradient of 5-20% of
N-(2-(benzyloxy)ethyl)-2-chloro-5-methoxyaniline (18). Commercially
available 9 (11.7 g, 60.5 mmol, 1 eq) was converted to the free base with
1M aqueous sodium carbonate (300 mL). The mixture was extracted with
CH2Cl2 (2×200 mL), the organic layer dried (MgSO4) and evaporated to
give an oil. 9 was then dissolved in CH2Cl2 (50 mL), in a dry flask under
nitrogen. Benzyloxyacetaldehyde 17 (9.36 mL, 10.0 g, 66.6 mmol, 1.1 eq,
d = 1.07 g/cm3) and acetic acid (3.81 mL, 4.0 g, 66. 6 mmol, 1.1 eq, d =
1.05 g/cm3) were added. After 15 minutes sodium triacetoxyborohydride
(19.3 g, 90.8 mmol, 1.5 eq) was added. The mixture was stirred for 18 h
at RT and then poured into saturated aqueous ammonium chloride
solution (100 mL) and extracted with CH2Cl2 (2×100 mL). The combined
organic layers were dried (MgSO4) and evaporated to give an oil. The
crude product was purified via silica flash chromatography eluting with
CH2Cl2 to yield 18 as an oil (13.0 g, 44.8 mmol, 74%). Material could not
be obtained in a pure form, but regardless reaction was progressed onto
a next step. LC-MS: Rf 2.42 (+ESI) m/z 292.3 ([M + H]+). 1H NMR (300
MHz, CDCl3): δH 7.37-7.30 (5H, m, Ph), 7.14 (1H, d, J = 8 Hz,
CHC(OCH3)CHCH), 6.23-6.18 (2H, m, CHC(OCH3)CHCH), 4.71 (2H, s,
OCH2Ph), 3.74 (3H, s, OCH3), 3.73 (2H, t, J = 5 Hz, CH2O), and 3.35 (2H,
dd, J = 6 Hz, CH2N). The compound was in complete agreement with
internal GE publication.
EtOAc/petroleum ether (40-60°C) to afford 13 as a yellow oil (1.1 g, 3.12
mmol, 31% over two steps). Material could not be obtained in a pure form,
but regardless reaction was progressed onto a next step. LC-MS: Rf 2.38
(+ESI) m/z 354.4 ([M + H]+). 1H NMR (300 MHz, CDCl3): δH 6.95 (1H, d, J
= 8 Hz, 7-CH), 6.35 (1H, d, J = 8 Hz, 6-CH), 4.90-4.40 (4H, m,
NCH2CH2F, NCH2CH2F), 4.20-4.10 (3H, m, 4-CH, CO2CH2CH3), 2.80-
2.65 (2H, m, 1-CH2), 3.79 (3H, s, OCH3), 2.10-1.80 (4H, m, 2- and 3-
CH2), and 1.30-1.20 (3H, m, CO2CH2CH3). The compound was in
complete agreement with internal GE publication.
Ethyl
9-(2-fluoroethyl)-5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-
carboxylate (14). The chloro intermediate 13 (1.10 g, 3.11 mmol, 1 eq)
was dissolved in methanol (50 mL) and triethylamine (0.517 mL, 377 mg,
3.73 mmol, 1.2 eq, d = 0.73 g/cm3) and 10% Pd/C (0.414 g) were added.
The mixture was stirred for 18 h after purging twice (over 2 h) with
hydrogen gas under atmospheric pressure. The reaction was filtered
through
a pad of celite under nitrogen atmosphere, washed with
methanol (50 mL), and the solvent was removed in vacuo. The residue
was dissolved in EtOAc (100 mL) and washed with 10% aqueous
potassium carbonate (100 mL), dried (MgSO4) and concentrated in
vacuo to give 14 as an oil (0.75 g, 2.35 mmol, 76%). LC-MS: Rf 2.18
(+ESI) m/z 320.5 ([M + H]+, 43%), Rf 2.20 (-ESI) m/z 318.4 ([M - H]-). H
1
Ethyl
3-((2-(benzyloxy)ethyl)(2-chloro-5-methoxyphenyl)amino)-2-
NMR (300 MHz, CDCl3): δH 7.04 (1H, t, J = 8 Hz, 7-CH), 6.84 (1H, d, J =
8 Hz, 8-CH), 6.46 (1H, d, J = 8 Hz, 6-CH), 4.64 (2H, dm, J = 47 Hz,
NCH2CH2F), 4.40-4.00 (5H, m, 4-CH, CO2CH2CH3, NCH2CH2F), 3.82
(3H, s, OCH3), 2.90-2.60 (2H, m, 1-CH2), 2.20-1.80 (4H, m, 2- and 3-
CH2), and 1.30-1.20 (3H, m, CO2CH2CH3). The compound was in
complete agreement with internal GE publication.
hydroxycyclohex-1-ene-1-carboxylate (19). The aniline 18 (14.1 g, 48.5
mmol, 1 eq) was stirred in dry THF (150 mL) at -40°C under nitrogen and
potassium bis(trimethylsilyl) amide (0.5 M solution in toluene, 203 mL,
20.2 g, 101.9 mmol, 2.1 eq) was added over 30 minutes. The carboxylate
11 (12.0 g, 48.5 mmol, 1 eq) in dry THF (50 mL) was then added and
allowed to warm to RT over a period of 1.5 h. Acetic acid was added (15
mL) and concentrated in vacuo to remove the THF. EtOAc (200 mL) and
10% aqueous potassium carbonate (200 mL) was added and the mixture
vigorously shaken. The EtOAc solution was separated, dried over
(MgSO4) and concentrated in vacuo to afford 19 as an oil (24.0 g, 52.2
mmol), which was used crude in the next step. LC-MS: Rf 2.39 (-ESI) m/z
458.5 ([M - H]-), Rf 2.53 (+ESI) m/z 460.5 ([M + H]+), Rf 2.39 (+ESI) m/z
460.5 ([M + H]+). The compound was in complete agreement with internal
GE publication.
9-(2-fluoroethyl)-5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylic
acid (15). The ester 14 (0.747 g, 2.34 mmol, 1 eq) was dissolved in
ethanol (10 mL). A solution of sodium hydroxide (1.13 g, 28.2 mmol, 12
eq) dissolved in 10 mL of water, was added. The reaction mixture was
heated to reflux for 18 h. The solvent was removed in vacuo and the
crude mixture diluted with water (30 mL), acidified with 2M HCl dropwise
until acidic, and washed with CH2Cl2 (50 mL). The organic layers were
combined and dried (MgSO4) and concentrated in vacuo to give 15 (0.66
g, 2.27 mmol), which was used crude into the next step. LC-MS: Rf 1.81
(+ESI) m/z 292.4 ([M + H]+).
Ethyl
9-(2-(benzyloxy)ethyl)-8-chloro-5-methoxy-2,3,4,9-tetrahydro-1H-
carbazole-4-carboxylate (20). Zinc chloride (19.6 g, 144 mmol, 3 eq) was
added to carboxylate 19 (22.0 g, 48.0 mmol, 1 eq) in dry Et2O (400 mL)
under nitrogen and heated at reflux for 5.5 h. As the reaction was
refluxed, a thick brown dense oil formed in the reaction. The reaction was
then cooled and the supernatant Et2O decanted off, EtOAc (300 mL) was
added, washed with 2M HCl (150 mL) and with 10% aqueous potassium
carbonate (150 mL). The EtOAc layer was separated, dried (MgSO4) and
concentrated in vacuo to afford an oil. The crude material was purified by
9-(2-fluoroethyl)-5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carbonyl
chloride (16). A solution of 15 (0.660 g, 2.27 mmol, 1 eq) in anhydrous
CH2Cl2 (5 mL) was stirred under nitrogen. Oxalyl chloride (0.584 mL, 865
mg, 6.81 mmol, 3 eq, d = 1.48 g/cm3) was added followed by a drop of
DMF. The reaction mixture was stirred at RT under nitrogen for 2 h then
evaporated in vacuo to give 16 (1.03 g, 3.33 mmol) as an oil, which was
used into the next step without purification.
silica gel chromatography eluting with
a
gradient of 10-40%
EtOAc/petroleum ether (40-60°C) to afford 20 as an oil (1.98 g, 4.49
mmol). The thick dense brown layer was treated with EtOAc (300 mL)
and 2M HCl (150 mL). The EtOAc layer was separated, washed with
10% aqueous potassium carbonate (150 mL), dried (MgSO4) and
concentrated in vacuo to give an oil. Et2O (400 mL) and anhydrous zinc
chloride (19.6 g, 144 mmol, 3 eq) were added. The mixture was heated
at reflux for a further 5 days. The Et2O layer and the dark gum were both
diluted with EtOAc (200 mL) and then washed with 2M HCl (150 mL),
dried (MgSO4) and concentrated in vacuo to give a gum. This gum was
purified by silica gel chromatography eluting with a gradient of 5-35%
EtOAc/petroleum ether (40-60°C) to afford 20 as an oil (16.1 g, 36.5
mmol, 85% total over 2 steps). LC-MS: Rf 2.66 (+ESI) m/z 442.5 ([M +
H]+). 1H NMR (300 MHz, CDCl3): δH 7.31-7.16 (5H, m, Ph), 6.93 (1H, d, J
= 8 Hz, 7-CH2), 6.34 (1H, d, J = 8 Hz, 6-CH2), 4.58 (2H, dm, J = 62 Hz,
NCH2CH2O), 4.40 (2H, s, OCH2Ph), 4.20-4.09 (3H, m, CO2CH2CH3 and
4-CH), 3.86-3.80 (2H, m, CH2OBn), 3.79 (3H, s, OCH3), 2.82-2.63 (2H, m,
N-benzyl-N-ethyl-9-(2-fluoroethyl)-5-methoxy-2,3,4,9-tetrahydro-1H-
carbazole-4-carboxamide (GE387) (6). The acid chloride 16 (1.03 g, 3.33
mmol, 1 eq) was dissolved in anhydrous CH2Cl2 (8 mL) and cooled to
0°C. N-ethylbenzylamine (0.977 mL, 901 mg, 6.67 mmol, 2 eq, d = 0.92
g/cm3) was then added and the reaction was stirred for 18 h at RT. The
reaction was quenched with 10% aqueous potassium carbonate (6 mL).
The CH2Cl2 layer was collected via a separatory funnel, dried (MgSO4),
and then concentrated in vacuo. The crude material was purified by silica
gel chromatography eluting with a gradient of 50-100% EtOAc/petroleum
ether (40-60°C) to afford the crude product as a yellow oil. The oil was
then recrystallized from EtOAc to afford 6 as a pale yellow solid (350 mg,
0.86 mmol, 26% over two steps). LC-MS: Rf 2.30 (+ESI) m/z 409.6 ([M +
H]+, 31%), Rf 2.35 (+ESI) m/z 409.6 ([M + H]+) . 1H NMR (300 MHz,
CDCl3): δH 7.42-7.21 (5H, m, Ph), 7.02 (1H, t, J = 8 Hz, 7-CH), 6.84 (1H,
d, J = 8 Hz, 8-CH), 6.43 (1H, d, J = 8 Hz, 7-CH), 4.80-4.40 (3H, m,
8
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10.1002/cmdc.201900023
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FULL PAPER
1-CH2), 2.10-1.80 (4H, m, 2- and 3-CH2), and 1.23 (3H, t, J = 7 Hz,
CO2CH2CH3). The compound was in complete agreement with internal
GE publication.
Dimethylaminopyridine (15.0 mg, 0.123 mmol, 0.1 eq) was then added
and the solution was stirred and warmed to RT for 18 h. The reaction
was then quenched by careful addition of ice followed by water (5 mL).
The mixture was then extracted into EtOAc and washed with water (3×10
mL). Excess pyridine was removed by washing with 1M HCl (2×10 mL)
and aqueous copper sulfate (2×10 mL). Excess 4-Toluenesulfonyl
chloride was removed by washing with 1M aqueous sodium carbonate
(2×10 mL). The organic layer was washed with brine (10 mL), dried
(MgSO4) and concentrated in vacuo. The crude product was
recrystallized from EtOAc to afford 25 as an off-white solid (220 mg, 0.40
mmol, 33%). LC-MS: Rf 2.46 (+ESI) m/z 561.6 ([M + H]+). 1H NMR (300
MHz, CDCl3): δH 7.51 ((2H, d, J = 8 Hz, SO2C(CH2)(CH2)CH3), 7.35-7.15
(5H, m, Ph), 7.09 (2H, d, J = 8 Hz, SO2C(CH2)(CH2)CH3), 6.91 (1H, t, J =
8 Hz, 7-CH), 6.62 (1H, d, J = 8 Hz, 8-CH), 6.37 (1H, d, J = 8 Hz, 6-CH),
4.70-4.50 (2H, m, CH2N), 4.30-4.15 (3H, m, 4-CH and CH2OH), 3.61 (3H,
s, OCH3), 3.80-3.40 (4H, m, N(CH2)2), 2.80-2.50 (2H, m, 1-CH2), 2.33
(3H, s, SO2PhCH3), 2.1-1.60 (4H, m, 2- and 3-CH2), and 1.30 (3H, t, J =
7 Hz, NCH2CH3).
9-(2-(benzyloxy)ethyl)-8-chloro-5-methoxy-2,3,4,9-tetrahydro-1H-
carbazole-4-carboxylic acid (21). To the ester 20 (0.401 g, 0.909 mmol, 1
eq) in ethanol (12 mL) was added sodium hydroxide (0.240 g, 6.00 mmol,
6.6 eq) in water (1 mL) and heated at 80°C for 18 h. The ethanol was
then removed by evaporation in vacuo and the residue partitioned
between Et2O (30 mL) and water (30 mL). The Et2O layer was separated,
dried (MgSO4) and concentrated in vacuo to give a gum. The aqueous
layer was acidified to pH 1 with 2M HCl dropwise and extracted with
CH2Cl2 (3×20 mL). The organic layers were dried (MgSO4) and
concentrated in vacuo to afford 21 as a solid (0.36 g, 0.87 mmol, 93%).
LC-MS: Rf 2.34 (+ESI) m/z 414.4 ([M + H]+). 1H NMR (300 MHz, CDCl3):
δH 7.31-7.15 (5H, m, Ph), 6.97 (1H, d, J = 8 Hz, 7-CH), 6.41 (1H, d, J = 8
Hz, 6-CH), 4.68-4.52 (2H, m, NCH2CH2O), 4.40 (2H, s, OCH2Ph), 4.18
(1H, t, J = 3 Hz, 4-CH), 3.88 (3H, s, OCH3), 3.82 (2H, t, J = 6 Hz,
CH2OBn), 2.86-2.63 (2H, m, 1-CH2), and 2.27-1.86 (4H, m, 2- and 3-
CH2). The compound was in complete agreement with internal GE
publication.
2-(4-(benzyl(ethyl)carbamoyl)-5-methoxy-1,2,3,4-tetrahydro-9H-carbazol-
9-yl)ethyl methanesulfonate (26). The phenol 24 (400 mg, 0.985 mmol, 1
eq) in CH2Cl2 (20 mL) was cooled to 0°C and methanesulfonyl chloride
(0.229 mL, 338 mg, 2.95 mmol, 3 eq, d = 1.48 g/cm3) and triethylamine
(0.409 mL, 299 mg, 2.95 mmol, 3 eq, d = 0.73 g/cm3) were added and
allowed to warm to RT for 18 h. The reaction was diluted with CH2Cl2 (20
mL) washed with 5% aqueous potassium carbonate solution (40 mL).
The layers were separated. The combined organic layers were dried
(MgSO4) and concentrated in vacuo to give a gum. The crude material
was purified by silica gel flash chromatography eluting with a gradient of
50-100% EtOAc/petroleum ether (40-60°C) to afford 26 as an oil (142 mg,
0.29 mmol, 37%). LC-MS: Rf 2.12 (+ESI) m/z 485.5 ([M + H]+). 1H NMR
(300 MHz, CDCl3): δH 7.40-7.10 (5H, m, NCH2Ph), 7.03 (1H, t, J = 8 Hz,
7-CH), 6.86 (1H, d, J = 8 Hz, 8-CH), 6.44 (1H, d, J = 8 Hz, 6-CH), 4.65-
4.50 (2H, m, CH2N), 4.50-4.45 (3H, m, 4-CH and CH2OH), 3.64 (3H, s,
OCH3), 3.90-3.20 (4H, m, N(CH2)2), 2.90-2.60 (2H, m, 1-CH2), 2.51 (3H,
s, OSO2CH3), 2.30-1.70 (4H, m, 2- and 3-CH2), and 1.31 (3H, t, J = 7 Hz,
NCH2CH3).
N-benzyl-9-(2-(benzyloxy)ethyl)-8-chloro-N-ethyl-5-methoxy-2,3,4,9-
tetrahydro-1H-carbazole-4-carboxamide (22). The acid 21 (2.23 g, 5.40
mmol, 1 eq) was dissolved in THF (135 mL) under N2 at RT. The solution
was allowed to cool to 0°C and N-ethylbenzylamine (0.870 mL, 803 mg,
5.94 mmol, 1.1 eq,
d =
0.92 g/cm3) was added followed by 1-
Hydroxybenzotriazole hydrate (0.802 g, 5.94 mmol, 1.1 eq) and N-(3-
Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (2.07 g, 10.8
mmol, 2 eq). Finally, triethylamine (2.99 mL, 2.18 g, 10.8 mmol, 4 eq, d =
0.73 g/cm3) was added via syringe and the mixture stirred under N2 and
warmed to RT over 48 h. The reaction was then diluted with EtOAc (100
mL) and filtered through celite. The celite was washed with more EtOAc
(2×100 mL). The combined filtrates were washed with 1M aqueous HCl
(2×150 mL), H2O (2×150 mL), brine (150 mL), dried (MgSO4) and
concentrated in vacuo to give the product 22 as an oil (1.55 g, 2.92 mmol,
54%), which was used crude in the next step. LC-MS: Rf 2.74 (+ESI) m/z
531.6 ([M + H]+).
(1R,2S,5R)-2-isopropyl-5-methylcyclohexyl
9-(2-(benzyloxy)ethyl)-8-
chloro-5-methoxy-2,3,4,9-tetrahydro-1H-carbazole-4-carboxylate (29). To
a stirring solution of 21 (300 mg, 0.726 mmol, 1 eq) and L-menthol in
CH2Cl2 (40 mL) at 0°C was added N-(3-Dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride (139 mg, 0.726 mmol, 1 eq) and 4-
N-benzyl-9-(2-(benzyloxy)ethyl)-N-ethyl-5-methoxy-2,3,4,9-tetrahydro-
1H-carbazole-4-carboxamide (23). The carboxamide 22 (1.50 g, 2.83
mmol, 1 eq) in methanol (120 mL) was shaken with 10% palladium on
charcoal (1.5 g), triethylamine (0.549 mL, 401 mg, 3.96 mmol, 1.4 eq, d =
0.73 g/cm3) and stirred for 18 h at RT after purging twice (over 2 h) with
hydrogen gas under atmospheric pressure. The reaction was then filtered
through a pad of celite under nitrogen atmosphere and the filtrate
concentrated in vacuo. The concentrate was then taken up in CH2Cl2
(200 mL) and washed with 5% aqueous potassium carbonate solution
(200 mL). The CH2Cl2 solution was then separated, dried (MgSO4) and
concentrated in vacuo to afford 23 as an oil (1.10 g, 2.22 mmol), which
was used crude in the next step. LC-MS: Rf 2.62 (+ESI) m/z 497.5 ([M +
H]+).
Dimethylaminopyridine (92.8 mg, 0.726 mmol,
1 eq). Stirring was
continued at RT for 16 h. After this period, H2O (100 mL) and CH2Cl2
(100 mL) were added. The organic layer was washed with 1M HCl,
saturated NaHCO3 solution, and brine. The solution was dried (MgSO4),
filtered, and concentrated. The crude racemic mixture 29 (360 mg, 0.65
mmol) was separated into two diasteriomerically enriched samples via
silica flash chromatography eluting with 30% Et2O/cyclohexane. LC-MS:
1
Rf 3.12 (+ESI) m/z 368.3 ([M -182]+, 100%). H NMR (300 MHz, CDCl3):
δH 7.36-7.12 (5H, m, Ph), 6.94 (1H, d, J = 8 Hz, 7-CH), 6.31 (1H, d, J = 8
Hz, 6-CH), 4.90 (1H, dt, J = 4 and 6 Hz, OCH), 4.38 (2H, d, J = 6 Hz,
NCH2CH2O), 4.09 (1H, t, J = 6 Hz, 4-CH), 3.86-3.77 (2H, m, CH2OBn),
3.75 (3H, s, OCH3), 2.7--2.44 (3H, m, 1-CH2, OCHCH2), 2.15-2.01 (1H, m,
OCHCH2), 2.00-1.82 (3H, m, 2-CH2, OCHCH), 1.81-1.56 (4H, m, 3-CH2,
CH3CH, OCHCHCH2), 1.46-1.40 (12H, m, CH3CHCH2, OCHCHCH2,
CHCH3, CH(CH3)2, and 1.02-0.83 (1H, m, CH3CHCH2).
N-benzyl-N-ethyl-9-(2-hydroxyethyl)-5-methoxy-2,3,4,9-tetrahydro-1H-
carbazole-4-carboxamide (24). The benzyl protected intermediate 23
(1.10 g, 2.22 mmol, 1 eq) in methanol (50 mL) was shaken with 10%
palladium on charcoal (0.333 g) and stirred for 18 h at RT after purging
twice (over 2 h) with hydrogen gas under atmospheric pressure. The
reaction was then filtered through
a pad of celite under nitrogen
(R)-1-phenylprop-2-yn-1-yl
2,3,4,9-tetrahydro-1H-carbazole-4-carboxylate (30). To a solution of 21
(300 mg, 0.73 mmol, eq), N-(3-Dimethylaminopropyl)-N′-
9-(2-(benzyloxy)ethyl)-8-chloro-5-methoxy-
atmosphere and the filtrate concentrated in vacuo to give 24 as an oil
(900 mg, 2.22 mmol), which was used crude in the next step. LC-MS: Rf
2.08 (+ESI) m/z 407.5 ([M + H]+).
1
ethylcarbodiimide hydrochloride (170 mg, 1.09 mmol, 1.5 eq) and 4-
Dimethylaminopyridine (6.96 mg, 0.04 mmol, 0.05 eq) in CH2Cl2 (5
mL)was added (R)-1-phenyl-2-propyn-1-ol (0.09 mL, 95.9 mg, 0.726
mmol, 1 eq, d = 1.07 g/cm3) at RT. The reaction was stirred for 4 h at RT.
The reaction mixture was diluted with CH2Cl2 (20 mL) and washed with
H2O (5 mL). The organic layer was dried (MgSO4) and concentrated. The
2-(4-(benzyl(ethyl)carbamoyl)-5-methoxy-1,2,3,4-tetrahydro-9H-carbazol-
9-yl)ethyl4-methylbenzenesulfonate (25). The alcohol 24 (500 mg, 1.23
mmol, 1 eq) was dissolved in anhydrous pyridine (5 mL) under N2. The
solution was cooled to 0°C and 4-Toluenesulfonyl chloride (515 mg, 2.71
mmol, 2.2 eq) added potion-wise to the solution over 30 minutes. 4-
9
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10.1002/cmdc.201900023
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crude mixture was purified via silica flash column chromatography eluting
with a gradient of 20-40% Et2O/petroleum ether (40-60°C) to yield 30 as
The final yield is 82.7 mg of enantiomer (S)-25 with 97.42% chemical
purity and in 98.2% ee, and 77.1 mg of enantiomer (R)-25 with 98.75%
chemical purity and in 99.6% ee. The specific stereochemistry of the two
enantiomers was not determined.
a
racemate (119 mg, 0.23 mmol, 31%). Separation of the two
diastereomer was done via silica column flash chromatography eluting
with a gradient of 10-20% Et2O/cyclohexane. LC-MS: Rf 2.72 (+ESI) m/z
528.5 ([M + H]+, 28%), 2.73 (+ESI) m/z 528.5 ([M + H]+, 51%). 1H NMR
(300 MHz, CDCl3): δH 7.60-7.14 (10H, m, Ph, OCHPh), 6.90 (1H, d, J = 8
Hz, 7-CH), 6.25 (1H, d, J = 8 Hz, 6-CH), 4.71-4.53 (2H, m, NCH2CH2O),
4.39 (1H, s, OCHPh), 4.15 (1H, t, J = 6 Hz, 4-CH), 3.81 (2H, t, J = 6 Hz,
CH2OBn), 3.40 (3H, s, OCH3), 2.85-2.51 (2H, m, 1-CH2), 2.60 (1H, d, J =
2 Hz, CCH), and 2.20-1.79 (4H, m, 2- and 3-CH2).
Determination of compound binding affinities at wild type and Ala147Thr
TSPO.
Binding affinities were measured as per our published protocol.[21] Briefly,
an Ultra-Turrax homogenizer was used to prepare membranes from HEK
cells stably transfected with WT and A147T TSPO. These cells have
been previously validated as an in vitro model of LABs and HABs.[21]
Membranes (20 ug per well, diluted in 50 mM Tris HCl, pH 7.4) were
incubated with ~ Kd concentration of [3H]1 (10 nM; Perkin Elmer) and test
compounds (0.3 nM – 10 µM) at 4 ºC for 90 min. Reactions were
terminated by filtration through a 96-well glass-fibre filter plate (Millipore),
and washed 8 times with ice-cold 50 mM Tris HCl. Microscint 0 was
added to the dry filters, and radioactivity read in a Microbeta2 2450
Microplate Counter (Perkin Elmer). Data were analysed using Graphpad
Prism 6.0 (GraphPad) and a four-parameter non-linear regression curve
fit was used to calculate the Ki values. Data are expressed as mean ±
SEM from at least three independent experiments.
Chiral Separation
N-benzyl-N-ethyl-9-(2-fluoroethyl)-5-methoxy-2,3,4,9-tetrahydro-1H-
carbazole-4-carboxamide (6) 49.8 mg of a racemic mixture of 6 was sent
to Reach Separations to be separated into the two enantiomers. They
provided the following methods for separation and purification. The
racemate 6 was dissolved to 12 mg/mL in MeOH:CH2Cl2 (1:1) and was
then purified by SFC using a Lux C1 column (21.2 mm x 250 mm, 5 µm)
at 40 °C and eluting with isocratic 50:50 MeOH:CO2 with 0.2% v/v NH3
with a flow rate of 50 mL/min. The injection volume was 1500 µL and
peaks were detected at 223 nm. Combined fractions of each of the two
enantiomers were then evaporated to near dryness using a rotary
evaporator, transferred into final vessels with CH2Cl2, which was
removed under a stream of compressed air at 40 °C before being stored
in a vacuum oven at 40 °C and 5 mbar for 4 h to afford the two
enantiomers as yellow gums. Chiral purity of the enantiomers was
analysed using a Lux C1 column (4.6 mm x 250 mm, 5 µm) at 40 °C and
eluting with isocratic 50:50 MeOH:CO2 with 0.2% v/v NH3 with a flow rate
of 4 mL/min. The injection volume was 1.0 µL and the peaks were
detected at 210-400 nm. Chemical purity of the enantiomers was
analysed using an Acquity BEH C18 column (50 x 2.1 mm, 1.7 µm) at
40 °C and eluting with 0-4 min gradient of 5-95% MeCN/H2O with 0.1%
TFA, 4-4.02 min gradient of 95-100% MeCN/H2O with 0.1% TFA, 4.03
min-4.5 min isocratic 100% MeCN with 0.1% TFA, 4.5-4.52 min gradient
of 100-5% MeCN/H2O with 0.1% TFA, and 4.53-6 min isocratic of 5%
MeCN/H2O with 0.1% TFA at flow rate 0.6 mL/min. The injection volume
was 1.0 µL and the peak was detected at 220 nm. The final yield is 23. 9
mg of enantiomer (S)-6 with 98.02% chemical purity and in 100% ee, and
22.1 mg of enantiomer (R)-6 with 100% chemical purity and in 98.8% ee.
The specific stereochemistry of the two enantiomers was not determined.
Circular dichroism spectroscopy. Solutions of (S)-6, (R)-6 and (S)-5 were
prepared in concentrations of 0.1-0.2 mg/mL in acetonitrile and CD
spectra collected on Aviv 410.
Radiochemistry
Radioisotope Production. No-carrier-added aqueous [18F]fluoride ion was
produced on a GE PETtrace cyclotron by irradiation of an 2.3-mL silver-
bodied water target with a 25-mA current and a 16.5-MeV proton beam
on 95% enriched 18O-H2O via the nuclear 18O(p, n)18F reaction.
Preparation of [18F]KF-Kryptofix-222 Complex. [18F]Fluoride in [18O]H2O
was transferred and immediately trapped on an anion-exchange resin
(Waters Sep-Pak Accell Light QMA cartridge in the carbonate form)
under vacuum. Trapped 18F-fluoride was eluted from the Sep-Pak
cartridge and transferred to the reaction vessel with an eluent solution
containing 0.25% wt Kryptofix-222® solution (1 mL) in basic (0.05% wt
K2CO3) aq MeCN (75% vv). The solvents were evaporated in vacuo (130
mbar) with a stream of N2 gas at 95 °C over 5 min. Anhydrous MeCN (3 x
0.7 mL) was then added and the mixture was azeotropically dried in
vacuo (130 mbar) with a stream of N2 at 95 °C.
2-(4-(benzyl(ethyl)carbamoyl)-5-methoxy-1,2,3,4-tetrahydro-9H-carbazol-
9-yl)ethyl 4-methylbenzenesulfonate (25) 162.4 mg of a racemic mixture
of 25 was sent to Reach Separations to be separated into the two
enantiomers. They provided the following methods for separation and
purification. The racemate 25 was dissolved to 23 mg/mL in
MeOH:CH2Cl2 (1:1) and was then purified by SFC using a Lux C1 column
(21.2 mm x 250 mm, 5 µm) at 40 °C and eluting with isocratic 50:50
MeOH:CO2 with 0.2% v/v NH3 with a flow rate of 50 mL/min. The
injection volume was 1000 µL and peaks were detected at 223 nm.
Combined fractions of each of the two enantiomers were then
evaporated to near dryness using a rotary evaporator, transferred into
final vessels with CH2Cl2, which was removed under a stream of
compressed air at 40 °C before being stored in a vacuum oven at 40 °C
and 5 mbar for 4 h to afford the two enantiomers as white solids. Chiral
purity of the enantiomers was analysed using a Lux C1 column (4.6 mm
x 250 mm, 5 µm) at 40 °C and eluting with isocratic 50:50 MeOH:CO2
with 0.2% v/v NH3 with a flow rate of 4 mL/min. The injection volume was
1.0 µL and the peaks were detected at 210-400 nm. Chemical purity of
the enantiomers was analysed using an Acquity BEH C18 column (50 x
2.1 mm, 1.7 µm) at 40 °C and eluting with 0-4 min gradient of 5-95%
MeCN/H2O with 0.1% TFA, 4-4.02 min gradient of 95-100% MeCN/H2O
with 0.1% TFA, 4.03 min-4.5 min isocratic 100% MeCN with 0.1% TFA,
4.5-4.52 min gradient of 100-5% MeCN/H2O with 0.1% TFA, and 4.53-6
min isocratic of 5% MeCN/H2O with 0.1% TFA at flow rate 0.6 mL/min.
The injection volume was 1.0 µL and the peak was detected at 220 nm.
Manual Preparation of [18F]6. Fluoride was dried as described above and
dissolved in MeCN (4mL) before eluting from TRACERlab FXFN. The
resulting solution was divided into three portions, two of which the MeCN
was removed at 100°C under a flow of N2 before dissolving in DMF or
DMSO. Precursors 25 and 26 were dissolved to a concentration of
1mg/mL in MeCN, DMSO and DMF to afford three solutions of each
precursor. For each reaction, four HPLC vials were prepared containing
precursor solution (125 µL) and
[
18F]fluoride solution (125 µL) of
equivalent solvent. The vials were heated in a heating block to the
desired temperature (80, 100 or 120°C) and a single vial was removed at
each time point (2, 5, 10, 20 min) and quenched with 1:1 MeCN/H2O
(300 µL) before analyzing directly by HPLC using a gradient method:
(pre-injection) 1 min equilibration at 10:90 MeCN/H2O. (post-injection) 1
min isocratic at 10:90 MeCN/H2O, 2 min gradient to 70:30 MeCN/H2O, 3
min isocratic at 70:30 MeCN/H2O. Flow rate: 1.5 mL/min. Run time: 6 min
(7 min including equilibration time). Column: ACE® UltraCore, Super C18,
2.5 µm, 50 x 4.6 mm. Serial No. A133239. A portion of the sample from
20 min was taken and used for co-injection with reference compound 6 to
confirm synthesis of desired product.
Automated Preparation and Formulation of [18F]6. The tosylate precursor
25 (1.2 mg, 2.94 µmol) was dissolved in anhydrous MeCN (1 mL), and
10
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10.1002/cmdc.201900023
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the solution was added to the dry [18F]KF-Kryptofix-222 complex and
heated at 100°C for 20 min. The reaction mixture was then cooled with
compressed air and diluted with anhydrous MeCN (1 mL). The crude
mixture was injected into an ACE 160433 C18 5-µm (100 x 10 mm) semi-
preparative reversed-phase HPLC column. With a mobile phase of 48%
aq MeCN at a flow rate of 5 mL/min. [18F]6 was collected (retention time:
19.6 min) and immediately diluted with H2O (10 mL). The aqueous
solution was passed through a C18 cartridge (Waters Sep-Pak Accell
Light C18 cartridge, prepared by washing with 2 mL of ethanol, rinsing
with 10 mL of water). The cartridge was washed with H2O (15 mL) and
the radiolabelled product eluted with EtOH (0.5 mL). The radiolabelled
product was then formulated with 0.9% w/v aq. NaCl in a vial to afford the
radiolabelled title compound.
PET. Whole brain TACs were then obtained for each of the animals. The
results were expressed as dimensionless standardized uptake values
(SUV
= (tissue activity x body weight)/injected dose). SUV were
calculated, assuming a specific gravity of 1 g/mL for brain tissue.
Acknowledgements
This work was supported by Medical Research Council (UK)
grant awards RG46503 (LM, MH, XZ) and RG70550 (EF),
National Institute of Health Research (UK), Cambridge
Biomedical Research Unit in Dementia (EF, NKR) and the
Herchel Smith Fellowship programme (LQ). The authors would
like to acknowledge Mr. Paul Burke and Dr. Roberto Canales for
their assistance with running the cyclotron. EPSRC National
Mass Spectrometry Facility at the University of Swansea is
acknowledged for performing accurate mass analysis. Dr.
Kathryn Stott (Biophysics facility) is acknowledged for
assistance with CD experiments. Dr Donna Lai and Dr Sheng
Hua are acknowledged for assistance with equipment in the
University of Sydney Molecular Biology Facility.
Quality Control for [18F]6. To confirm the identity of the formulated
radiotracer, and for the determination of molar radioactivity and
radiochemical purity,
a portion of the final solution with known
radioactivity was injected into a Phenomenex Luna 5µ C18 (2) 100Å (250
x 4.6 mm) analytic reversed-phase HPLC column. A mobile phase of 0-3
min isocratic at 10% aq. MeCN, 3-5 min gradient 10-80% aq. MeCN, and
6-12 min isocratic 80% aq. MeCN at a flow rate of 1.5 mL/min to elute
[
18F]6 at a tR of 9.8 min. The area of the UV absorbance peak at 220 nm
corresponding to the carrier product was measured (integrated) on the
HPLC chromatogram and compared with a standard curve relating mass
to UV absorbance. The identity of the radiolabelled product was
confirmed by HPLC co-injection of the reference compound 6.
Keywords: TSPO receptor • PET imaging • Neuroinflammation
biomarker • [18F]GE180 • [18F]GE387
In Vivo PET Imaging
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Entry for the Table of Contents
20
15
SS
U
U
V
V
10
5
0
0
3.5
2.0
O
N
O
0
210
230
250
270
290
*
-5
N
-10
-15
-20
-25
-30
18F
[
18F]6
[18F]GE387
(Sies1)-5 (GE180)
Ser
Series2
(S)-6 (GE387)
sagittal
transversal
coronal
Series3
(R)-6 (GE387)
Wavelength (nm)
A novel PET radiotracer candidate GE387 for imaging TSPO expression associated with neuroinflammation was studied. The
synthesis of reference material and radiolabelling precursor as well as the manual and automated radiosynthetic methods are
presented. Separation into enantiomers was achieved using the SFC and absolute configuration determined through CD comparison
to GE180. PET candidate showed promising initial in vivo PET data.
13
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