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. 2019 Apr 4;10(5):743–748. doi: 10.1021/acsmedchemlett.8b00643

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 Patché †, Bryan Veeren †, Sébastien Bénard ‡, Fanny Gimié ‡, Jennyfer Yong-Sang †, Ilya Khantalin §, Reuben Veerapen ∥, Emmanuelle Jestin †,‡, Olivier Meilhac †,§,*
PMCID: PMC6511949  PMID: 31097993

Abstract

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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 potential risk prediction factor of plaque rupture. Darapladib is one of the most potent Lp-PLA2 inhibitors with an IC50 of 0.25 nM. Using its affinity for Lp-PLA2, we describe herein the total synthesis of darapladib radiolabeling precursor and the automated radiolabeling process for positron emission tomography (PET) imaging via an arylboronate moiety. The tracer thus obtained was tested in a mouse model of atherosclerosis (ApoE KO) and compared with the widely used [18F]fluorodeoxyglucose ([18F]FDG) PET tracer, known to label metabolically active cells. [18F]Darapladib showed a significant accumulation within mice aortic atheromatous plaques dissected out ex vivo compared to [18F]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.

Keywords: Atherosclerosis, darapladib, PET imaging, Lp-PLA2, automated radiofluorination, aryl boronate

In western countries, cardiovascular diseases and in particular atherosclerotic complications are responsible for about 50% of death.1 Atherosclerosis is characterized by an accumulation of lipids and inflammatory cells within the vascular wall of large arteries.2 In spite of important therapeutic progresses, fatal events due to atherosclerotic diseases remain a growing public health issue. Increasing efforts are made to find new noninvasive imaging tools to assess plaque localization, composition, and susceptibility to rupture.3 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 including cancer,4 neurological,5 or heart diseases.6 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 resolution imaging, and covalent radiolabeling.7,8 In nuclear medicine, [18F]fluorodeoxyglucose ([18F]FDG) is the most successful radiopharmaceutical used for PET diagnosis based on the increased metabolic demand of cells, particularly in oncology. However, [18F]FDG use for detection of atherosclerosis presents some limitations due to the heterogeneity of glucose consumption by vascular and inflammatory cells.9 There is an important need for identification of vulnerable plaques, prone to rupture. In atherosclerotic plaques, lipoprotein-associated phospholipase A2 (Lp-PLA2) is a well-established biomarker associated with 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 with its ability to efficiently hydrolyze oxidized phospholipids, leading to the formation of lysophosphatidylcholine and oxidized free fatty acids.11 Lp-PLA2 activity and presence could represent useful biomarkers to identify cardiovascular diseases and offer a potent therapeutic alternative to known methods through Lp-PLA2 inhibition.12 This enzyme has been targeted by pharmaceutical groups in order to develop specific inhibitors.13−16 Darapladib was developed by GSK as the most potent Lp-PLA2 inhibitor (IC50 = 0.25 nM, using human recombinant enzyme).17 Since critical end points 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.18−20 Taking advantage of the strong affinity of darapladib for Lp-PLA2,21,22 the demonstrated in vivo safety, and the presence of a fluorine in its structure, we used [18F]darapladib to target atherosclerotic plaques by PET imaging.

Since the original structure of darapladib (Chart 1) is preserved, both physicochemical properties (solubility, polarity, etc.) and in vivo behavior (metabolism, catabolism, etc.) of the radioactive molecule will be similar to the native compound. As shown in Chart 1, in order to 18F-radiolabel darapladib on the aryl position of group 2, we chose to synthesize an aryl-boronate precursor. SNAr reactions are the most widely used methods to produce 18F-labeled arenes. There are two main strategies available to perform radiofluorination of arenes, devoid of strong electron-withdrawing groups.23 The first involves the radiofluorination of strong electrophiles such as iodonium ylides24 or diaryliodonium salts.25 However, with electron neutral arenes, these reactions require high temperature and offer low radiochemical yields (RCY).24 To address these limitations, a second strategy was initiated by Scott et al.26 They proposed, under mild conditions, the use of K18F to obtain 18F-labeled electron-rich, neutral, and deficient aryl fluorides. Shortly after, Gouverneur et al.27,28 offered a new pathway that targets direct nucleophilic 18F– fluorination of a wide range of arylboronate esters with K18F/K222 mediated by the commercially available copper complex [Cu(OTf)2(Py)4]. Scott et al. then proposed an alternative that directly forms, in situ, the copper complex.29 This method allows more constant radiochemical yields and radiofluorination of both boronates and boronic acid derivatives. We report herein the synthesis of 18F-radiolabeled darapladib via alcohol-enhanced Cu-mediated radiofluorination30 (Scheme 1) in order to image atherosclerotic 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 4) were obtained as previously described.32

Chart 1. Four Moieties of Darapladib Involved in Lp-PLA2 Recognitiona.

Chart 1

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a Groups 1 and 2 are mainly involved in binding to Lp-PLA2 catalytic site. Red arrow: targeted site for 18F-radiolabeling.

Scheme 1. Copper-Mediated Radiofluorination of Aryl Boronates.

Scheme 1

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(1) Scott et al. pathway with formation of the copper complex in situ. (2) Zlatopolskiy et al. alcohol-enhanced radiofluorination with 30% alcohol charge. (3) Radiolabeling strategy for [18F]darapladib consisting in both in situ complex formation and 30% alcohol charge.

We first synthesized the arylboronate moiety that will allow 18F-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 bromination via CBr4 to obtain 3 in 94% yield. As shown in Scheme 3, we then synthesized the radiolabeling boronate precursor 9 of darapladib. Thiouracile 4 was synthesized as previously described32 and engaged in a substitution with alkylating agent 3 to afford compound 5 in 98% yield.

Scheme 2. Synthesis of Boronate Derivative 3.

Scheme 2

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Reagents and conditions: (a) NaBH4, THF/MeOH 7/3, 0° C for 30 min; (b) KOAc, bis(pinacolato)diboron, Pd(dppf)Cl2, DMSO, 85 °C for 18 h; (c) CBr4, PPh3, THF, RT for 18 h.

Scheme 3. Synthesis of the Radiolabeling Precursor 9.

Scheme 3

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Reagents and conditions: (a) KI, K2CO3, 3, acetone, 54°C for 3 h; (b) tert-butyl-bromoacetate, DIPEA, DCM, 40°C for 20 h; (c) TFA, TIS, DCM, RT for 20 h; (d) COMU, DIPEA, DMF, 0°C for 1 h then RT for 12 h.

Nonselective 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)32 and 7 allowed us to obtain darapladib-arylboronate radiolabeling precursor 9 in 70% yield.

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 18F– from anion exchanger QMA cartridge was performed using a KOTf/K2CO3 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.29 Reactions were carried out at 110 °C under vacuum for 20 min. Gouverneur et al. explain that the copper complex might need to be reoxidized 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.

Scheme 4. Radiosynthesis of [18F]Darapladib from 9.

Scheme 4

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Table 1. Copper-Mediated Radiofluorination of 9.

entry solvents activity (MBq)a RCYb
1 DMF 257 <1%
2 NMP 379 1%
3 NMP/n-BuOHc 2310 6%
4 NMP/n-BuOHc 1979 5%
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a

Before formulation process.

b

Decay corrected.

c

Starting activity: 58 000 MBq (entry 3), 52 000 MBq (entry 4).

In 2017, Zlatopolskiy et al. proposed an alcohol-enhanced copper-mediated radiofluorination that replaced water azeotropic removal by eluting Et4NHCO3 in n-BuOH of QMA-trapped 18F–. They also showed that the use of NMP offered a better yield than DMF with 30% alcohol charge. In our hands, 18F– 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 18F– yield (up to 95%). Compound 9 was dissolved in a 990 μL mixture of NMP/n-BuOH (0.6/0.3; v/v) and was added to the reactor containing previously dried 18F–. This protocol allowed us to increase [18F]darapladib radiofluorination up to 6% (entries 3 and 4). Automated radiofluorination RCY obtained were similar or even better than those reported in the literature for automated copper-mediated 18F-radiofluorination. The final solution concentrations were around 200 ± 50 MBq/mL, which is suitable for preclinical studies requirements.33

[18F]Darapladib purity (>99%) and specific activity (40–60 GBq/μmol) were assessed by radio-HPLC (see Supporting Information). Radiolabeled darapladib retention time was similar to the cold reference [19F]darapladib and no radioactive contaminants were detected. [18F]Darapladib stability (see Supporting Information) was confirmed in saline buffer at 0, 30, and 90 min with no degradation observed. In order to evaluate the ability of [18F]darapladib to target atherosclerotic plaques, 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. [18F]Darapladib was compared to [18F]FDG, which was suggested to label inflammation associated with atherosclerotic plaques.34 Syringes (15 ± 5 MBq) of [18F]darapladib or [18F]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 allow us to localize [18F]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 nothing was observable in the aorta. [18F]Darapladib accumulated mainly in the liver, kidneys, and intestines, suggesting a binding to LDL/HDL-associated Lp-PLA2 since these organs are involved in lipoprotein metabolism.33 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 was already explained by Dave and co-workers.35

In comparison, [18F]FDG accumulated in high-glucose consuming organs (brain/heart) and in kidneys and bladder, as they are involved in glucose catabolism. To evaluate [18F]darapladib accumulation within atheromatous vessels, the heart and aorta were dissected out 15 min postinjection, 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 [18F]darapladib or [18F]FDG in C57BL/6 mice (below detection threshold), devoid of plaques (Figure 1). In contrast, a strong labeling of the aorta was observed in ApoE KO mice with [18F]darapladib (4.6 ± 0.8%ID/g) but not with [18F]FDG (below detection threshold). This suggests that darapladib may represent a good radiotracer for detection of atheromatous plaques. These results were comforted by immunohistochemistry staining showing the high presence of Lp-PLA2 within mice aortas and aortic sinuses (see Supporting Information). In order to consolidate these results, we tested the ability of [18F]darapladib to label human carotid atherosclerotic samples known to exhibit high level of Lp-PLA2. Both [18F]darapladib and [18F]FDG were tested ex vivo in freshly obtained carotid endarterectomy samples (Figure 2). Our previously reported study showed that excised tissue is still alive and metabolically active.36 As depicted in Figure 2, [18F]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 noncomplicated plaques). More interestingly, [18F]darapladib was able to significantly accumulate in both complicated and noncomplicated parts of atherosclerotic carotid samples ten times higher than [18F]FDG (0.1%ID/g for both culprit and noncomplicated plaques). This highlights that, using our 18F-labeled ligand, accumulation of Lp-PLA2 associated with lipoproteins and monocytes/macrophages may represent an interesting target for imaging subclinical atherosclerosis. Immunohistochemistry for detection of Lp-PLA2 was performed on the same samples (see Supporting Information), and a strong staining was observed on both complicated and noncomplicated parts, supporting the hypothesis that [18F]darapladib binds Lp-PLA2 in human endarterectomy samples. These results were supported by DESI-IMS studies that show association between the presence of [19F]darapladib and Lp-PLA2 after incubation of carotid samples with the cold compound (see Supporting Information). Since [18F]darapladib seems to be more effective than [18F]FDG for imaging inflammatory cells in atherosclerosis, we tested its ability to accumulate in tumors, also known to contain monocytes/macrophages.

Figure 1.

Figure 1

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Ex vivo aorta and heart PET imaging of [18F]darapladib and [18F]FDG in ApoE KO and C57BL/6 mice. 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, and 2D slice.

Figure 2.

Figure 2

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Ex vivo accumulation of [18F]darapladib compared to [18F]FDG in human carotid samples. (1) Macroscopic view (ncp, noncomplicated plaque; cp, culprit plaque 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 planes a and b.

Radiolabeled darapladib or [18F]FDG (15 ± 5 MBq) were injected via the caudal vein in melanoma B16-injected mice that develop tumors (see Supporting Information).34 As expected, [18F]FDG provided an intense radiolabeling of tumors, whereas [18F]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 [18F]darapladib than [18F]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 Lp-PLA2-expressing cells such as macrophages and lipoproteins provide good targets for [18F]darapladib.

We have described the synthesis of the radiolabeling precursor of darapladib. Through aryl-boronate pathway, we were able to perform copper-mediated 18F-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 studies performed with [18F]FDG and [18F]darapladib showed a good affinity of our labeled Lp-PLA2 ligand for atheromatous plaques relative to [18F]FDG, which was more specific for tumor detection. The suitability of [18F]darapladib for detection of Lp-PLA2 within atherosclerotic plaques was confirmed in human samples. Although RCY and specific activities may be improved, the use of [18F]darapladib for imaging in clinical settings could be of interest, in particular since phase I and II clinical trials reported darapladib use in humans.37 Further investigations would be needed to assess the specificity of [18F]darapladib in other preclinical models using bigger animals (i.e., rabbits or pigs) and to confirm its safety for imaging trials.

Acknowledgments

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).

Glossary

Abbreviations

COMU

(1-cyano-2-ethoxy-2-oxoethylidenaminooxy) dimethyl aminomorpholino-carbenium hexafluorophosphate

DCM

dichloromethane

DESI–IMS

desorption electrospray ionization–ion mobility spectrometry

DIPEA

N,N-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 methylammonium

Tf

triflate

TFA

trifluoroacetic acid

THF

tetrahydrofuran

TIS

triisopropyl silane

PET

positron emission tomography

RCY

radiochemical yield

Supporting Information Available

. The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmedchemlett.8b00643.

  • Chemistry, radiochemistry, and imaging experimental procedures; NMR spectra; FX-FN module, stabilities, and biodistribution studies; DESI-MS and IHC studies (PDF)

Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. The authors would like to thank Dr. Frédéric Dollé for fruitful discussions.

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. F.G. was supported by a CIFRE doctoral grant no. 2013/1522.

The authors declare no competing financial interest.

Supplementary Material

ml8b00643_si_001.pdf (1.9MB, pdf)

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