Synthesis and Automated Labeling of [18F]Darapladib, a Lp-PLA2 Ligand, as Potential PET Imaging Tool of Atherosclerosis

Article abstract of DOI:10.1021/acsmedchemlett.8b00643

Atherosclerosis and its associated clinical complications are major health issues in industrialized countries. Lipoprotein-associated phospholipase A₂ (Lp-PLA₂) was demonstrated to play an important role in atherogenesis and to be a

Full text of DOI:10.1021/acsmedchemlett.8b00643

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Letter
Synthesis and Automated Labeling of [18F]Darapladib, a Lp-
PLA2 ligand, as potential PET Imaging tool of Atherosclerosis
Florian Guibbal, Vincent Meneyrol, Imade Ait-Arsa, Nicolas Diotel, Jessica
Patche, Bryan Veeren, Sébastien Bénard, Fanny Gimie, Jennyfer Yong-Sang,
Ilya Khantalin, Reuben Veerapen, Emmanuelle Jestin, and Olivier Meilhac
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00643 • Publication Date (Web): 04 Apr 2019
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Synthesis and Automated Labeling of [ F]Darapladib, a Lp-PLA
ligand, as potential PET Imaging tool of Atherosclerosis
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Florian Guibbal , Vincent Meneyrol , Imade Ait-Arsa , Nicolas Diotel , Jessica Patché , Bryan Veeren ,
Sébastien Bénard , Fanny Gimié , Jennyfer Yong-Sang , Ilya Khantalin , Reuben Veerapen ,
2
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4
1
,2
1,3
Emmanuelle Jestin and Olivier Meilhac *
(
1) Université de La Réunion, INSERM, UMR 1188 Diabète athérothrombose Thérapies Réunion Océan Indien (DéTROI),
Saint-Denis de La Réunion, France
(
2) CYclotron Réunion Océan Indien CYROI, 2 rue Maxime Rivière, 97490 Sainte-Clotilde
3) CHU de La Réunion, Saint-Denis de La Réunion, France
(
(4) Clinique de Sainte-Clotilde, 127, Route de Bois de Nèfles, Sainte-Clotilde, Réunion, France
ABSTRACT: Atherosclerosis and its associated clinical complications are major health issues in industrialized countries. Lp-PLA
2
(
Lipoprotein-associated phospholipase A
prediction factor of plaque rupture. Darapladib is one of the most potent Lp-PLA
for Lp-PLA , we describe herein the total synthesis of darapladib radiolabeling precursor and the automated radiolabeling process for
PET imaging via an arylboronate moiety. The tracer thus obtained was tested in a mouse model of atherosclerosis (ApoE KO) and
2
) was demonstrated to play an important role in atherogenesis and to be a potential risk
2
inhibitors with an IC50 of 0.25 nM. Using its affinity
2
1
8
18
compared with the widely used [ F]FDG PET tracer, known to label metabolically active cells. [ F]Darapladib showed a significant
accumulation within mice aortic atheromatous plaques dissected out ex vivo compared to [ F]FDG. Incubation of the radiotracer
with human carotid samples showed a strong accumulation within the atherosclerotic plaques and supports its potential for use in
PET imaging.
18
KEYWORDS : Atherosclerosis, darapladib, PET imaging, Lp-PLA
2
, automated radiofluorination, aryl boronate
In western countries, cardiovascular diseases and in particular
atherosclerotic complications are responsible for about 50% of
death. Atherosclerosis is characterized by an accumulation of
leading to the formation of lysophosphatidylcholine and
oxidized free fatty acids. Lp-PLA activity and presence could
2
represent useful biomarkers to identify cardiovascular diseases
and offer a potent therapeutic alternative to known methods
through Lp-PLA
by pharmaceutical groups in order to develop specific
inhibitors.
most potent Lp-PLA
recombinant enzyme). Since critical endpoints were not met
(cardiovascular death, coronary events, myocardial infarction
and stroke) in both STABILITY and SOLID-TIMI 52 studies,
darapladib failed to pass phase III clinical trials.
advantage of the strong affinity of darapladib for Lp-PLA
the demonstrated in vivo safety and the presence of a fluorine in
1
1
1
lipids and inflammatory cells within the vascular wall of large
arteries. In spite of important therapeutic progresses, fatal
2
12
2
inhibition. This enzyme has been targeted
events due to atherosclerotic diseases remain a growing public
health issue. Increasing efforts are made to find new non-
invasive imaging tools to assess plaque localization,
1
3,14,15,16
Darapladib was developed by GSK as the
inhibitor (IC50 = 0.25 nM, using human
2
3
17
composition and susceptibility to rupture. Functional
molecular imaging can enable early detection of disease and
assessment of therapy efficacy. Positron emission tomography
(PET) has become a powerful tool to diagnose disorders
1
8,19,20
Taking
2
,
4
5
6
21,22
including cancer, neurological or heart diseases. Fluorine-18
is the most widely used radioisotope for PET diagnosis due to
its relatively short half-life (t1/2 = 109.8 min), high spatial
1
8
its structure, we used [ F]darapladib to target atherosclerotic
plaques by PET imaging.
7
,8
resolution imaging and covalent radiolabeling. In nuclear
1
8
18
medicine, [ F]fluorodeoxyglucose or [ F]FDG is the most
successful radiopharmaceutical used for PET diagnosis based
on the increased metabolic demand of cells, particularly in
Chart 1. Four moieties of darapladib involved in Lp-PLA
recognition. Groups 1 and 2 are mainly involved in binding
to Lp-PLA
2
1
8
2
catalytic site. Red arrow: targeted site for F-
1
8
oncology. However, [ F]FDG use for detection of
atherosclerosis presents some limitations due to the
heterogeneity of glucose consumption by vascular and
radiolabeling.
O
N
Group 1
F
N
9
F
F
Group 2
F
inflammatory cells. There is an important need for
S
N
identification of vulnerable plaques, prone to rupture. In
atherosclerotic plaques, lipoprotein-associated phospholipase
A (Lp-PLA ) is a well-established biomarker associated with
2 2
O
N
Group 3
the accumulation of both low-density lipoproteins (LDL) and
inflammatory cells such as macrophages.10 The pro-
inflammatory properties of this enzyme have been associated to
its ability to efficiently hydrolyze oxidized phospholipids,
Group 4
Since the original structure of darapladib (Chart 1) is
preserved, both physico-chemical properties (solubility,
polarity, etc.) and in vivo behavior (metabolism, catabolism,
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Scheme 2. Synthesis of boronate derivative 3.
1
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O
a
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9
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compound. As shown in Chart 1, in order to F-radiolabel
darapladib on the aryl position of group 2, we chose to
synthesize an aryl-boronate precursor. SNAr reactions are the
Br
Br
H
OH
1
1
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b
most widely used methods to produce F-labeled arenes. There
are two main strategies available to perform radiofluorination
of arenes, devoid of strong electron-withdrawing groups. The
first involves the radiofluorination of strong electrophiles such
as iodonium ylides or diaryliodonium salts. However, with
electron neutral arenes, these reactions require high temperature
and offer low radiochemical yields (RCY). To address these
limitations, a second strategy was initiated by Scott et al. They
proposed, under mild conditions, the use of K F to obtain F-
labeled electron-rich, neutral, and deficient aryl fluorides.
offered a new pathway that
targets direct nucleophilic F fluorination of a wide range of
O
c
O
2
3
B
B
O
Br
O
OH
3
2
2
4
25
4
Reagents and conditions: (a) NaBH , THF/MeOH 7/3, 0°C for 30 min; (b)
KOAc, bis(pinacolato)diboron, Pd(dppf)Cl
CBr , PPh , THF, RT for 18h.
2
, DMSO, 85°C for 18h; (c)
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1
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Non-selective alkylation with tert-butyl-bromoacetate yielded
the desired N1-ester 6 (26%) which was then hydrolyzed with
TFA using a carbocation scavenger (TIS) to afford acid 7
(64%). Peptide coupling of 8 (previously described) and 7
allowed us to obtain darapladib-arylboronate radiolabeling
precursor 9 in 70% yield.
2
7,28
Shortly after, Gouverneur et al.
3
2
1
8
-
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arylboronate esters with K F/K222 mediated by the
commercially available copper complex [Cu(OTf) (Py) ]. Scott
et al. then proposed an alternative that directly forms, in situ,
2
4
Scheme 3. Synthesis of the radiolabeling precursor 9.
2
9
the copper complex. This method allows more constant
radiochemical yields and radiofluorination of both boronates
and boronic acid derivatives. We report herein the synthesis of
F-radiolabeled darapladib via alcohol-enhanced Cu-mediated
radiofluorination (Scheme 1) in order to image atherosclerotic
O
O
O
a
N
b
N
NH
N
H
4
S
N
H
S
N
S
18
O
O
O
5
B
6
B
30
O
O
O
plaques. K222 was excluded from our automated method since
its use was demonstrated to be detrimental to reaction yields.31
We used our previously reported darapladib synthesis method
in order to produce the radiofluorination precursor. Darapladib
biphenyl moiety and aliphatic chain (respectively groups 3 and
c
O
N
F
F
F
N
HN
+
3
2
8
N
S
4
) were obtained as previously described.
O
O
7
B
Scheme 1. Copper-mediated radiofluorination of aryl
boronates.
OH
O
d
O
F
N
F
F
N
S
N
O
O
B
N
O
9
2 3
Reagents and conditions: (a) KI, K CO , 3, acetone, 54°C for 3h; (b) tert-
butyl-bromoacetate, DIPEA, DCM, 40°C for 20h; (c) TFA, TIS, DCM, RT
for 20h; (d) COMU, DIPEA, DMF, 0°C for 1h then RT for 12h.
The precursor 9 was then engaged in a copper-mediated aryl-
radiofluorination using a GE Healthcare TRACERlab FX FN
module (Scheme 4). Initially, we worked under Scott et al.
conditions in order to produce radiolabeled darapladib. Elution
of F- from anion exchanger QMA cartridge was performed
using a KOTf/K CO aqueous solution (5 mg/50 µg in 550 µL).
The eluted fluorine was azeotropically dried with acetonitrile
60°C then 120°C) since copper-mediated labeling showed
water sensitivity. Reactions were carried out at 110°C under
vacuum for 20 min. Gouverneur et al. explain that the copper
complex might need to be re-oxidized since highly reactive
copper(III) species may be responsible for the SNAr reaction.
Thus, air was injected 4 times during the reaction course since
very low yields (<1%) were obtained without air addition. The
reaction mixture was then loaded on preparative HPLC for
purification after passing through a Sep-Pak Alumina N
cartridge and then formulated in a 5% EtOH saline solution. As
presented in Table 1, radiofluorination in DMF produced the
desired radiotracer in a very low yield (entry 1) and NMP did
not provide any significant improvement (entry 2), both
methods leading to a maximum of 1% recovery.
1
8
2
3
(
(1) Scott et al. pathway with formation of the copper complex in situ. (2)
29
Zlatopolskiy et al. alcohol-enhanced radiofluorination with 30% alcohol
1
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charge. (3) Radiolabeling strategy for [ F]darapladib consisting in both in
situ complex formation and 30% alcohol charge.
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We first synthetized the arylboronate moiety that will allow F-
labeling (Scheme 2). 4-bromobenzaldehyde was reduced to the
corresponding alcohol 1 in 91% yield. In a palladium-catalyzed
coupling, the boronate moiety was inserted in 91% followed by
4
bromination via CBr to obtain 3 in 94% yield. As shown in
Scheme 3, we then synthetized the radiolabeling boronate
precursor 9 of darapladib. Thiouracile 4 was synthetized as
3
2
previously described and engaged in a substitution with
alkylating agent 3 to afford compound 5 in 98% yield.
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nothing was observable in the aorta. [18F]Darapladib
accumulated mainly in the liver, kidneys and intestines,
Scheme 4. Radiosynthesis of [ F]darapladib from 9.
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3
4
5
6
7
8
9
O
Cu(OTf)2 / Py
O
2
suggesting a binding to LDL/HDL-associated Lp-PLA , since
K18
air
F
33
N
N
these organs are involved in lipoprotein metabolism. These
results were supported by immunohistochemistry staining with
anti-Lp-PLA2 antibody (see supporting information) showing
the presence of our target in livers. Accumulation within
kidneys and intestines were already explained by Dave and co-
N
S
N
S
NMP / n-BuOH
10°C 20 min
O
O
O
1
18F
B
R
O
R
R = 8
35
workers.
In 2017, Zlatopolskiy et al. proposed an alcohol-enhanced
copper-mediated radiofluorination that replaced water
azeotropic removal by eluting Et NHCO in n-BuOH of QMA-
4 3
trapped F . They also showed that the use of NMP offered a
better yield than DMF with 30% alcohol charge. In our hands,
F elution using n-BuOH did not offer sufficient yields (less
than 20% of the starting activity). We thus kept our eluting
conditions, which offered a better F yield (up to 95%).
Compound 9 was dissolved in a 990 L mixture of NMP/n-
BuOH (0.6/0.3; v/v) and was added to the reactor containing
Figure 1. Ex vivo aorta and heart PET imaging of
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[
F]darapladib and [ F]FDG in ApoE KO and C57BL/6
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-
mice.
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8 -
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8 -
1
8 -
previously dried F . This protocol allowed us to increase
F]darapladib radiofluorination up to 6% (entries 3 and 4).
18
[
Automated radiofluorination RCY obtained were similar or
even better than those reported in the literature for automated
18
copper-mediated F-radiofluorination. The final solutions
concentrations were around 200 ± 50 MBq/mL that are suitable
3
3
for preclinical studies requirements.
Table 1. Copper-mediated radiofluorination of 9.
Entry
Solvents
Activity
RCY[b]
[
a]
(MBq)
The heart was removed for dissection of the aorta; its position is indicated
with an orange dashed circle. From left to right: macroscopic picture, 3D
view, 2D slice.
1
2
3
4
DMF
257
<1%
1%
NMP
379
In comparison, [18F]FDG accumulated in high-glucose
NMP / n-BuOH[c]
NMP / n-BuOH[c]
2310
1979
6%
consuming organs (brain/heart) and in kidneys and bladder, as
they are involved in glucose catabolism. To evaluate
5%
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[ F]darapladib accumulation within atheromatous vessels, the
heart and aorta were dissected out 15 min post-injection,
flushed with saline and imaged ex vivo (15 min PET
acquisition) (Figure 1). The aortas were then opened
longitudinally, split and pined onto a black wax surface, in order
to expose the atheromatous plaques. Ex vivo PET images were
compared to macroscopic captures of atheromatous plaques. No
accumulation was observed in the aorta for either
[
5
a] Before formulation process; [b] Decay corrected; [c] Starting activity:
8 000 MBq (entry 3), 52 000 MBq (entry 4).
1
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[ F]Darapladib purity (>99%) and specific activity (40-60
GBq/µmol) were assessed by radio-HPLC (see supporting
information). Radiolabeled darapladib retention time was
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similar to the cold reference [ F]darapladib and no radioactive
1
8
contaminants were detected. [ F]Darapladib stability (see
supporting information) was confirmed in saline buffer at 0, 30
and 90 min with no degradation observed. In order to evaluate
18
18
[
F]darapladib or [ F]FDG in C57BL/6 mice (below detection
threshold), devoid of plaques (Figure 1). In contrast, a strong
18
labeling of the aorta was observed in ApoE KO mice with
the ability of [ F]darapladib to target atherosclerotic plaques,
1
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18
[ F]darapladib (4.6 ± 0.8 %ID/g) but not with [ F]FDG (below
detection threshold). This suggests that darapladib may
represent a good radiotracer for detection of atheromatous
the radiotracer was injected in a mouse model known to develop
aortic atheroma plaques (Apolipoprotein E- deficient or ApoE
KO mice) and compared to wild type C57BL/6 mice.
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plaques.
These
results
were
comforted
by
[ F]Darapladib was compared to [ F]FDG, which was
suggested to label inflammation associated with atherosclerotic
immunohistochemistry staining showing the high presence of
3
4
18
18
Lp-PLA within mice aortas and aortic sinuses (see supporting
plaques. 15 ± 5 MBq syringes of [ F]darapladib or [ F]FDG
were injected via the caudal vein in 10 month-old ApoE KO and
C57BL/6 mice. Since no differences were observed between 15
and 45 min acquisitions, the optimal imaging time was set at 15
min for subsequent experiments. Whole body imaging did not
2
information). In order to consolidate these results, we tested the
1
8
ability of [ F]darapladib to label human carotid atherosclerotic
samples known to exhibit high level of Lp-PLA . Both
F]darapladib and [ F]FDG were tested ex vivo in freshly
2
18
18
[
18
obtained carotid endarterectomy samples (Figure 2). Our
allow us to localize [ F]darapladib in the aorta, due to the high
uptake by other organs such as liver (19 ± 2 %ID/g), intestines
32 ± 1 %ID/g) and kidneys (21 ± 1 %ID/g) (see supporting
information). Injection in C57BL/6 wild type mice led to a
similar uptake in these major organs liver (23 ± 3 %ID/g),
intestines (26 ± 5 %ID/g) and kidneys (11 ± 4 %ID/g) but
previously reported study showed that excised tissue is still
3
6
alive and metabolically active. As depicted in Figure 2,
(
1
8
[
F]FDG-incubated carotid samples displayed a weak signal
only in the stenosed complicated plaque sample (0.02 %ID/g
for culprit and 0.01 %ID/g for non-complicated plaques). More
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interestingly, [ F]darapladib was able to significantly
accumulate in both complicated and non-complicated parts of
plaques relative to [ F]FDG that was more specific for tumor
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detection. The suitability of [ F]darapladib for detection of Lp-
PLA within atherosclerotic plaques was confirmed in human
samples. Although RCY and specific activities may be
improved, the use of [ F]darapladib for imaging in clinical
settings could be of interest, in particular since phase I and II
clinical trials reported darapladib use in Humans. Further
investigations would be needed to assess the specificity of
[ F]Darapladib in other preclinical models using bigger
animals (i.e rabbits or pigs) and to confirm its safety for imaging
trials.
1
8
atherosclerotic carotid samples ten times higher than [ F]FDG
(0.1 %ID/g for both culprit and non-complicated plaques). This
highlights that, using our F-labeled ligand, accumulation of
Lp-PLA associated with lipoproteins and
monocytes/macrophages may represent an interesting target for
imaging sub-clinical atherosclerosis. Immunohistochemistry
for detection of Lp-PLA
see supporting information) and a strong staining was observed
on both complicated and non-complicated parts, supporting the
hypothesis that [ F]darapladib binds Lp-PLA in human
2
endarterectomy samples. These results were supported by
DESI-IMS studies that show association between the presence
of [ F]darapladib and Lp-PLA
samples with the cold compound (see supporting information).
2
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was performed on the same samples
(
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AUTHOR INFORMATIONS
Corresponding Author
1
9
2
after incubation of carotid
Phone: +(262) 262 938 811. E-mail: olivier.meilhac@inserm.fr
1
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18
Since [ F]darapladib seems to be more effective than [ F]FDG
for imaging inflammatory cells in atherosclerosis, we tested its
ability to accumulate in tumors, also known to contain
monocytes/macrophages.
Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the
manuscript.
1
8
Figure 2. Ex vivo accumulation of [ F]darapladib
Funding Sources
1
8
compared to [ F]FDG in human carotid samples.
This work was supported by the ERDF (European Regional
Development Fund) 33054 and RE0001897 (EU- Région Réunion
-French State national counterpart) as well as a Philancia fund.
Florian Guibbal was supported by a CIFRE doctoral grant
n°2013/1522.
[18
F]Darapladib
1
2
3a
ncp
3b
ncp
ncp
cp
Acknowledgments
cp
cp
This work has benefited from the facilities and expertise of the
Small Molecule Mass Spectrometry platform of ICSN and NMR
service (Centre de Recherche de Gif-www.icsn.cnrs-gif.fr).
a
b
[18
F]FDG
1
2
ncp
3
Abbreviations
ncp
ncp
cp
COMU ((1-Cyano-2-ethoxy-2-oxoethylidenaminooxy) dimethyl
cp
cp
aminomorpholino-carbenium
(dichloromethane), DESI-IMS (desorption electrospray ionization
ion mobility spectrometry), DIPEA (N,N-
hexafluorophosphate),
DCM
-
1
) Macroscopic view (ncp: non-complicated plaque, cp: culprit plaque
Diisopropylethylamine), DMA (dimethylacetamide), DMF (N,N-
dimethylformamide), DMSO (dimethyl sulfoxide), FDG
(Fluorodeoxyglucose), GSK (Glaxo Smith Kline), HDL (high
density lipoprotein), LDL (low density lipoprotein), NMP (N-
methyl-2-pyrrolidone), Py (pyridine), QMA (quaternary methyl
ammonium), Tf (triflate), TFA (trifluoro acetic acid), THF
(tetrahydrofuran), TIS (triisopropyl silane), PET (positron emission
tomography), RCY (radiochemical yield).
corresponding to the stenosed part of the same carotid sample; 2) 3D PET
imaging view showing slice planes a and b in orange; 3) corresponding
orthoslice of plans a and b.
1
8
Radiolabeled darapladib or [ F]FDG (15 ± 5 MBq) were
injected via the caudal vein in melanoma B16-injected mice that
develop tumors (see supporting information). As expected,
F]FDG provided an intense radiolabeling of tumors whereas
F]darapladib imaging was surprisingly negative in this model
its accumulation was observed only in the liver, kidney and
intestines). These results suggest a better specificity of
F]darapladib than [ F]FDG for detection of atheromatous
plaques. The cells composing the plaque may have a lower
glucose uptake than cancer cells. In contrast, the presence of
-expressing cells such as macrophages and
lipoproteins provide good targets for [ F]darapladib.
We have described the synthesis of the radiolabeling precursor
of darapladib. Through aryl-boronate pathway, we were able to
perform copper-mediated F-labeling of darapladib on an
automated module (n = 10) with good radiochemical purity
assessed by radio-HPLC (>99%) and specific activity (40-60
GBq/µmol). Although the sensitivity of micro-PET did not
allow us to detect small atheromatous plaques in vivo, PET
3
4
1
8
[
[
(
1
8
Supporting information
Chemistry, radiochemistry and imaging experimental procedures,
NMR spectra. FX-FN module, stabilities and biodistribution
studies. DESI-MS and IHC studies.
18
18
[
Lp-PLA
2
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4-week study to evaluate the effect of rilapladib on cognition and
2
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