# Synthesis, labeling, and biological evaluation of P2Y 12 receptor radioligands for positron emission tomography imaging of neuroinflammation

Eugénie Pincemail, Marie-Anne Peyronneau, Caroline Denis, Hayet Bensalah, Margot Corbel, Sébastien Goutal, Maud Goislard, Haneen Al Hroub, Marie Théry, Sébastien Beuché, et al.

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## ► To cite this version:

Eugénie Pincemail, Marie-Anne Peyronneau, Caroline Denis, Hayet Bensalah, Margot Corbel, et al. Synthesis, labeling, and biological evaluation of P2Y 12 receptor radioligands for positron emission tomography imaging of neuroinflammation. ACS Chemical Neuroscience, 2025, 16 (14), pp.2639-2656. 10.1021/acschemneuro.5c00187. hal-05136822

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Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
Synthesis, labeling and biological evaluation of
P2Y₁₂ receptor radioligands for positron emission
tomography imaging of neuroinflammation

Eugénie Pincemail,¹ Marie-Anne Peyronneau,¹ Caroline Denis,¹ Hayet Bensalah,¹ Margot Corbel,² Sébastien Goutal,¹ Maud Goislard,¹ Haneen Al Hroub,³ Marie Théry,¹ Sébastien Beuché,¹,† Hashem Ali M Al Musawi,³ Fabien Chauveau,⁴ Nadja Van Camp,² Alexis Bemelmans,² Christa E. Müller,³ Bertrand Kuhnast,¹ Alexandra Winkeler¹ and Mylène Richard*¹

¹Paris-Saclay University, CEA, CNRS, Inserm, BioMaps, SHFJ, 91401 Orsay, France.

²Paris-Saclay University, CEA, CNRS, MIRCen, Laboratoire des Maladies Neurodegeneratives,
92265 Fontenay-aux-Roses, France.

³PharmaCenter Bonn, Pharmaceutical Institute, Department of Pharmaceutical & Medicinal Chemistry, University of Bonn, 53121 Bonn, Germany.

⁴Université Claude Bernard Lyon 1, Centre de Recherche en Neurosciences de Lyon, Inserm U1028, CNRS UMR5292, BIORAN, Groupement Hospitalier Est, 69677 Bron, France

Corresponding author: Mylène Richard, mylene.richard@cea.fr
ABSTRACT

The P2Y₁₂ receptor (P2Y₁₂R) is a G protein-coupled receptor whose expression level is directly
correlated to microglial activation. Herein we report on the design of a series of new P2Y₁₂R
ligands and the radiolabeling and characterization of two positron emission tomography (PET)
tracers, [¹¹C]37 and [¹⁸F]41. These compounds were evaluated by autoradiography studies on rat
brain slices exhibiting overexpression of human P2Y₁₂Rs (AAV-hP2Y₁₂R). Metabolism and
biodistribution of [¹⁸F]41 were evaluated ex vivo in healthy rats and indicated good metabolic
stability with 41 % of unchanged radioligand 1 hour post injection and a limited crossing of the
blood-brain barrier with a brain uptake of 0.02%ID/g 1 hour post injection. In vivo PET imaging
performed in the AAV-hP2Y₁₂R rat model confirmed this low brain uptake and no significant
difference was found in the transfected (SUVmean 0.14 ± 0.01) versus contralateral (SUVmean 0.13
± 0.01) striatum of the AAV-hP2Y₁₂R model. Similar results were observed in healthy rats and in
non-human primates. Additional studies in the presence of tariquidar led to a 3-4-fold increase in
[¹⁸F]41 brain concentration, suggesting that [¹⁸F]41 is a P-glycoprotein substrate. Future work will
focus on improving radioligand design to enhance blood-brain barrier permeation and to reduce
efflux transport.

KEYWORDS

P2Y₁₂R, PET ligands, neuroinflammation, microglial state, fluorine-18, carbon-11
INTRODUCTION

Microglia are resident cells of the central nervous system (CNS) implicated in normal CNS function, maintenance of homeostasis and inflammatory processes including both injury and repair.¹ Microglial cells are present in different dynamic states, with variable morphology that correlate with transcriptional, metabolomic and proteomic profiles.¹,² Morphological modifications include changes from a ramified morphology to an amoeboid (i.e. without ramifications) state, often associated with brain injury or disease.³ The change in microglial morphology is associated to alterations in cell surface receptors expression, cytokine and chemokine secretion, and can be associated to a change in functional status of microglia.⁴⁻⁶

Positron Emission Tomography (PET) is now one of the main tools for imaging neuroinflammation. This highly sensitive and non-invasive imaging modality is well suited to explore the molecular processes and functional changes relevant to diseases responsible for neuroinflammation. To date, most of the development in PET imaging of neuroinflammation has been focused on targeting translocator protein 18 kDa (TSPO).⁷ However, although many radioligands have been developed, TSPO presents some limitations due to its polymorphism affecting affinity of the ligands⁸ and its expression in different brain cells (microglia, endothelial cells, astrocytes and neurons).⁹ Besides, even though TSPO-PET imaging has proven the implication of microglia in neurological pathologies, it does not distinguish between differential microglial states, previously referred to as pro- and anti-inflammatory. Given these drawbacks, alternative microglia-selective biomarkers are required to image changes in microglial activation status to allow for a follow-up during disease progression.¹⁰⁻¹² This pressing need is driving the development of PET tracers of microglial receptors associated to microglial status, which will ultimately guide therapeutic strategies aimed at modulating inflammation from injury to repair.
In 2017, Beaino *et al*. showed that P2Y₁₂ and P2X₇ receptors are relevant and selective targets for PET imaging to discriminate different microglial states in multiple sclerosis (MS).¹³ In particular, P2Y₁₂R, a G-protein coupled receptor expressed by microglia in the brain, was identified as a specific homeostatic marker.¹⁴ Increasing evidence indicates that P2Y₁₂R expression is strongly downregulated in disease-associated microglia according to the disease stage. A study from 2017 showed that the expression of P2Y₁₂R was correlated to the astrocytoma grade and to the activation state of microglia cells in samples of patient tumors.¹⁵ In a demyelination model of MS, a reduced expression of P2Y₁₂R was observed in active inflammatory lesions.⁵ Similarly, Zrzavy and coworkers observed a loss of P2Y₁₂R expression in active and expending lesions and an increased expression of the receptor in completely inactive lesions in which inflammation has subsided.¹⁶ Another study showed a decrease of microglial P2Y₁₂ receptor levels in a mouse model of tauopathy, despite an increase in the total microglial population.¹⁷ In light of these results, the P2Y₁₂ receptor appears to be a promising target to evaluate the changes in microglial homeostasis by PET imaging.

Due to its high expression levels in platelets and its important role in thrombosis, the P2Y₁₂ receptor has been widely studied, and potent and selective synthetic agonists and antagonists have been developed.¹⁸ To date, only a few P2Y₁₂R radiotracers have been reported and their suitability for PET imaging has been examined. In 2018, Villa *et al*.¹⁴ published the synthesis and evaluation of a high affinity P2Y₁₂R radioligand based on a tetrasubstituted pyridine, [¹¹C]5 (Figure 1 - A). However, in vivo experiments in healthy rats showed very limited crossing of the blood brain barrier (BBB) and poor metabolic stability. Maeda *et al*.¹⁷ and Jackson *et al*.¹⁹ reported a similar pyridine-based radioligand, [¹¹C]AZD1283 (Figure 1 - B). Although autoradiography experiments and metabolic stability were promising, only low brain uptake was observed in healthy rhesus
macaques and in wild-type mice with this radioligand. Around the same time, two
thienopyrimidine-based radioligands were published by Windhorst and coworkers (Figure 1 –
C).²⁰ While autoradiography studies showed high P2Y₁₂R targeting, brain uptake was low in
healthy Wistar rats and blocking of efflux transporters with tariquidar led to a strong increase of
the radioactivity in the brain, indicating efflux transport of the radioligand.

In the present study, we aimed to develop new P2Y₁₂R radioligands based on a lead compound previously described by Boldron and coworkers (Figure 1 – D).²¹ This ligand presents an IC₅₀ of 17 nM for the human P2Y₁₂R and its structure possesses a methylated amine, making isotopic labeling with carbon-11 a possible strategy. New fluorinated and methylated analogues were thus synthetized and their affinity for P2Y₁₂R and their metabolic behaviour were evaluated in order to select the most promising compounds. Radiolabeling of two of these analogues was then implemented and automated, and their in vivo biodistribution and metabolic stability were evaluated on healthy rats.

<figure><img src="image_3.png" /><figcaption>**Figure 1.** Current P2Y₁₂R radioligands (A-C) and ligand with potential isotopic labeling site (D).</figcaption></figure>
# RESULTS AND DISCUSSION

## Synthesis of methylated and fluorinated ligands

Reference ligands were prepared following previously reported work with some modifications.²¹ The synthesis relies on a convergent strategy requiring two key synths, an indole-carboxylic acid derivative and an aryl amine (Scheme 1). Several modifications of the lead compound structures were envisioned, substitution at the 5-position of the indole moiety (chlorine or methyl group), variation of one of the aromatic rings (phenyl or pyridazine) or different linkers between the indole moiety and the radioactive atom.

**Scheme 1.** General strategy for the synthesis of P2Y₁₂R ligands.

<figure><img src="image_4.png" /></figure>

Firstly, condensation of commercial chloroaryl-hydrazines **1** and **2** with enamine **4** gave the corresponding pyrazoles **5** and **6** (Scheme 2). Nucleophilic substitution of chlorine of compound **5** led to the protected aminopyridazine **7**, however the reaction was inefficient on **6** and compound **8** was accessed through a palladium-catalyzed Buchwald-Hartwig reaction. After hydrolysis of the ethyl ester, 4-carboxypyrazoles **9** and **10** were activated with carbonyldiimidazole (CDI) and reacted with 3-ethoxy-3-oxopropanoate magnesium salt and malonates **11** and **12** were then alkylated by reaction with ethyl iodide under basic conditions. The last step was the concomitant
decarboxylation and deprotection of the amine function in strong acidic conditions, leading to the
expected amines intermediates **15** and **16** in 17 and 51% yield over 6 steps.

<figure><img src="image_5.png" /><figcaption>**Scheme 2.** Synthesis of amines **15** and **16**.</figcaption></figure>

Reagents and conditions: a) *N,N*-dimethylformamide dimethyl acetal, room temperature (rt), 5 h (99%); b) EtOH, reflux, 2 h, **5** (44%), **6** (74%); c) for **7**: 4-methoxybenzylamine, 1,4-dioxane, reflux, 24 h (91%); for **8**: Pd(OAc)₂, (±)-binap, Cs₂CO₃, 4-methoxybenzylamine, THF, 100 °C, 2 days (92%); d) KOH, EtOH, H₂O, 80 °C, 2 h, **9** (99%), **10** (94%); e) (i) CDI, THF, rt, 12 h, (ii) MgCl₂, ethyl potassium malonate, 55 °C, 7 h, **11** (64%), **12** (85%); f) ethyl iodide, Cs₂CO₃, DMF, rt, 5 h, **13** (87%), **14** (94%); g) 37% HCl, reflux, 4 h, **15** (79%), **16** (100%).

Synthesis of functionalized carboxylic acids **27** and **28** started with the acylation of the 5-substitued indoles **17** and **18** with trifluoroacetic anhydride (Scheme 3). Carboxylic acids **21** and **22** were then obtained by hydrolysis of the trifluoroacetyl group. Preliminary experiments showed that protection of the carboxylic acid group prior to methylation of the indole was mandatory. We thus opted for benzyl ester as a protecting group, leading to compounds **23** and **24**. Alkylation of the indole moiety with methyl bromoacetate followed by careful hydrogenolysis of the benzyl protecting group with Pd/C under hydrogen atmosphere yielded the expected carboxylic acids **27** and **28** in 5 steps and in 41 and 45% yields, respectively.

<caption>**Scheme 3.** Synthesis of carboxylic acids **27** and **28**</caption>
<figure><img src="image_6.png" /></figure>

Reagents and conditions: a) (CF<sub>3</sub>CO)<sub>2</sub>O, Et<sub>2</sub>O, 0°C, 2 h, **19** (82%), **20** (83%); b) KOH, H<sub>2</sub>O, refluxed, 8 h, **21** (93%), **22** (78%); c) (i) (CF<sub>3</sub>CO)<sub>2</sub>O, CH<sub>2</sub>Cl<sub>2</sub>, rt, 2 h, (ii) BnOH, rt, 3 h, **23** (99%), **24** (83%); d) methyl bromoacetate, K<sub>2</sub>CO<sub>3</sub>, DMF, 80°C, 2 h, **25** (69%), **26** (87%); e) Pd/C (10%), H<sub>2</sub>, THF, rt, 2 h, **27** (78%), **28** (96%).

The coupling between carboxylic acids **27-28** and amines **15-16** was optimized on model molecules (see SI for full details of optimization) and the best conditions were employed for the synthesis of compounds **29-32** (Scheme 4). Briefly, coupling with pyridazine-amine **15** required activation of the carboxylic acid counterpart **27** or **28** with methanesulfonyl chloride and *N*-methyl imidazole prior to addition of amine and heating of the reaction mixture for several days in a sealed vial.<sup>22</sup> These harsh conditions were required due to the low nucleophilicity of the amine function induced by the pyridazine scaffold. Regarding aniline derivative **16**, classic coupling conditions (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 4-dimethylaminopyridine) were successfully employed for the coupling with **27** and **28**. The obtained methyl esters **29-32** were then hydrolyzed by reaction of sodium hydroxide in ethanol and the corresponding carboxylic acids **33-36** were coupled to the appropriate amines, leading to ligands **37-44** in yields ranging from 15 to 49% in 3 steps.

**Scheme 4.** Preparation of ligands **37-44**
<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

Reagents and conditions: a) for X = N, (i) NMI, MsCl, CH₂Cl₂, 0°C, 90 min; (ii) **27** or **28**, 50°C, sealed vial, 5 d, **29** (54%), **30** (44%); for X = CH, EDC, DMAP, CH₂Cl₂, 50°C, 4 h, **31** (61%), **32** (63%); b) 2 M NaOH, MeOH, rt, 6 h, **33** (89%), **34** (81%), **35** (100%), **36** (97%); c) amine, HATU, DIPEA, DMF, rt, 2 h, **37** (48%), **38** (43%), **39** (84%), **40** (69%), **41** (75%), **42** (43%), **43** (63%), **44** (35%).

## Biological characterization

The affinity of ligands **37-44** were determined via a competition binding assay using a novel tritiated radioligand [³H]PSB-22219²³ (Table 1, see structure in SI, section II). Regarding methylated references **38-40**, Ki varied from 10.4 to 12.1 nM, indicating no influence of the substituent at the 5-position of the indole ring and no influence of the central aromatic moiety (phenyl or pyridazine). A value of 43.8 nM was obtained for ligand **37**, similar to its IC₅₀ reported value (17 nM).²¹ Regarding fluorinated references **41-44**, similar affinities were found, with Ki
values ranging from 11.2 to 26.5 nM. Influence of the substituent at position 5 of the indole ring was also proven to be minimal, as seen with the comparable Kᵢ values obtained for compound **41** (5-chloro, Kᵢ = 17.1 ± 0.8 nM) and compound **42** (5-methyl, Kᵢ = 15.3 ± 2.4 nM). The PEGylated chain of compound **44** seems to slightly reduce its affinity for P2Y₁₂R, showing a Kᵢ value of 26.5 ± 3.4 nM.

<table><caption>**Table 1:** Kᵢ values of P2Y₁₂R ligands determined in radioligand competition binding assay versus [³H]PSB-22219.ª</caption><thead><tr><th></th><th>Compounds</th><th>X</th><th>Y</th><th>NRR'</th><th>Kᵢ ± SEM (nM)<sup>b</sup></th></tr></thead><tbody><tr><td rowspan="8"></td><td>37</td><td>N</td><td>Cl</td><td rowspan="4">N-N-<br/>N-<br/>N-<br/>N</td><td>43.8 ± 3.9<sup>c</sup></td></tr><tr><td>38</td><td>N</td><td>CH<sub>3</sub></td><td>11.5 ± 0.9</td></tr><tr><td>39</td><td>CH</td><td>Cl</td><td>12.1 ± 1.2</td></tr><tr><td>40</td><td>CH</td><td>CH<sub>3</sub></td><td>10.4 ± 1.6</td></tr><tr><td>41</td><td>CH</td><td>Cl</td><td rowspan="2">N-<br/>N-<br/>N-<br/>N</td><td>17.1 ± 0.8</td></tr><tr><td>42</td><td>CH</td><td>CH<sub>3</sub></td><td>15.3 ± 2.4</td></tr><tr><td>43</td><td>CH</td><td>CH<sub>3</sub></td><td>N-<br/>N-<br/>N-<br/>N</td><td>11.2 ± 1.3</td></tr><tr><td>44</td><td>CH</td><td>CH<sub>3</sub></td><td>N-<br/>N-<br/>N-<br/>N</td><td>26.5 ± 3.4</td></tr></tbody></table>

## In vitro metabolic studies on unlabeled ligands

Liquid chromatography/mass spectrometry (LC/MS) metabolic profiles of P2Y₁₂R ligands **37-44** were obtained by incubation of the parent ligands with rat liver microsomes (RLM) or human liver microsomes (HLM) in the presence of a NADPH-generating system. These incubations resulted in the formation of 3 to 5 main metabolites more polar than the parent compound (see full fragmentation tables and MS chromatograms in supplementary data, section III). LC/MS analyses and MS2 experiments suggest the formation of hydroxylated and dealkylated (N- and O-

<footnote>ª See recent publication²³, ᵇ Data represent mean ± SEM of 3-4 independent experiments each in duplicates, ᶜ The higher value is probably due to a solubility issue during the experiments.</footnote>
dealkylation) derivatives of the ligands. In rat liver microsomes, a low level of metabolism was observed for all compounds as 98 to 88% of the parent compounds remained intact after 60 minutes of incubation (Figure 2). HLM were similar or even less active than RLM, except in the case of compound 44, for which N- and O-dealkylation were observed. These reactions are known to be mainly catalyzed by CYP3A4 in human liver microsomes. In HLM, the main metabolite observed was due to O-dealkylation. A control reaction without NADPH-generating system did not produce any detectable metabolites after a 60 minutes incubation.

<figure><img src="image_9.png" /><figcaption>**Figure 2.** Percentages of parent ligands remaining after 60 minutes incubation with RLM (n = 4) or HLM (n = 2), independent experiments performed in duplicate. Values were expressed as mean % ± SD.</figcaption></figure>

Following MS/MS experiments, the structures of the metabolites could be proposed by comparison between fragmentation profiles of the molecular ions for each P2Y<sub>12</sub>R derivative and its metabolites (Figure 3, Table 2). The main MS/MS fragments for these compounds came from: (i) the cleavage of the butanone chain (loss of C<sub>4</sub>H<sub>7</sub>O•), (ii) the cleavage of the bond between the pyridazine or phenyl ring and the amide function, (iii) the cleavage of the bond between the position 3 of the indole moiety and the amide function (leading to C<sub>15</sub>H<sub>16</sub>N<sub>3</sub>O<sub>2</sub>• or C<sub>13</sub>H<sub>14</sub>N<sub>5</sub>O<sub>2</sub>• fragments), (iv) the cleavage of the second amide bond with the loss of the methyl-piperazine
group for **37-40** or the fluoroPEG group for **44** or the fluoro-ethyl piperidine group for **41** and **42**
or the fluoroethyl chain for **43**, (v) the cleavage between the amide bond and the nitrogen atom of
the indole ring. Furthermore, smaller fragments containing the indole moiety of the compounds
were also detected.

<figure><img src="image_10.png" /><figcaption>**Figure 3.** Fragmentations (A) and potential metabolites (B) of P2Y<sub>12</sub>R ligands</figcaption></figure>

The mass of the molecular ions of the first group of metabolites detected was 16 Da higher than
the molecular ion of the parent compounds, in agreement with the insertion of an oxygen atom
into the molecules by hydroxylation. One reaction of hydroxylation probably occurred at the
terminal position (methyl) of the butanone chain and another one occurred at the pyrazole-methyl
position. The third reaction of hydroxylation was likely to occur at the indole moiety of the
molecule (methyl or pyrrole ring), as suggested by the fragments containing the indole moiety of
the molecules 16 Da higher than the molecular ion of the parent fragments. For N-methylated
compounds **37-40**, the mass spectrum of the third metabolite exhibited a molecular ion 14 amu
lower than the molecular ion of the parent compounds, which is consistent with a reaction of N-
demethylation occurring at the methyl-piperazine group of the parent compounds. For **44**, the mass
spectrum of two metabolites exhibited molecular ions 46 and 90 amu lower than the molecular ion
of the parent compound, in agreement with the loss of the fluoroethyl chain (m/z 546) and the fluoroethoxy ethyl chain (m/z 502) by O-dealkylation. For **43**, the mass spectrum of the fourth metabolite exhibited a molecular ion 46 amu lower than the molecular ion of the parent compound, in agreement with the loss of the fluoroethyl chain by N-dealkylation. For **41** and **42**, the presence of the molecular ion with a m/z of -2 compared to the parent ion may result from an oxidative defluorination of the tracer.²⁴

<table><caption>**Table 2.** MS and MS/MS analysis of parent compounds and their main metabolites in RLM.</caption><thead><tr><th>compounds</th><th>37</th><th>38</th><th>39</th><th>40</th><th>41</th><th>42</th><th>43</th><th>44</th></tr></thead><tbody><tr><td>Parent ion (m/z)</td><td>563</td><td>543</td><td>561</td><td>541</td><td>592</td><td>572</td><td>504</td><td>592</td></tr><tr><td>Hydroxylation (+16)</td><td></td><td>559</td><td></td><td>557</td><td>608</td><td>588</td><td>520</td><td>608</td></tr><tr><td>Hydroxylation (+16)</td><td>559</td><td>559</td><td>577</td><td>557</td><td>608</td><td>588</td><td>520</td><td>608</td></tr><tr><td>Hydroxylation (+16)</td><td>559</td><td>559</td><td>577</td><td>557</td><td>608</td><td>588</td><td>520</td><td>608</td></tr><tr><td>Hydroxylation (+16)</td><td></td><td></td><td></td><td></td><td></td><td>588</td><td></td><td></td></tr><tr><td>Oxidative defluorination</td><td></td><td></td><td></td><td></td><td>590</td><td>570</td><td></td><td></td></tr><tr><td>N-demethylation (-14)</td><td>549</td><td>529</td><td>547</td><td>527</td><td></td><td></td><td></td><td></td></tr><tr><td>N/O-dealkylation</td><td></td><td></td><td></td><td></td><td></td><td></td><td>458,</td><td></td></tr><tr><td>(-134, -90, -46)</td><td></td><td></td><td></td><td></td><td></td><td></td><td>458</td><td>502,</td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>546</td></tr></tbody></table>

Taken together, these results indicate a good metabolic stability of the new P2Y₁₂R ligands, in RLM as well as in HLM. These in vitro results and the brain metabolism were further investigated in vivo with the radiolabeled ligands.

## Radiolabeling of P2Y₁₂R ligands

Based on the results of metabolism and affinity studies on unlabeled ligands, compounds **37** and **41** were selected for their better stability, good affinities and ease of synthesis. Radiolabeling of
37 and 41 with carbon-11 and fluorine-18, respectively, before in vitro and in vivo studies was then optimized. Radiolabeling with carbon-11 necessitated the preparation of non-methylated labeling precursor 46, obtained in two steps from carboxylic acid 33 (Scheme 5). Compound 33 was first coupled to Boc-protected piperazine and the protecting group was then cleaved by trifluoroacetic acid in dichloromethane, giving the expected precursor 46 in 55% yield. Similarly, labeling with fluorine-18 required the preparation of a precursor bearing a leaving group for nucleophilic substitution with the [<sup>18</sup>F]F<sup>-</sup> anion. To this end, tosylated compound 47 was prepared in one step from carboxylic acid 35 in 61% yield via coupling with 4-tosylethylpiperidine.

<figure><img src="image_11.png" /><figcaption>**Scheme 5.** Preparation of labeling precursors 46 and 47</figcaption></figure>

**Reagents and conditions:** a) 1-*N*-Boc-piperazine, HATU, DIPEA, DMF, rt, 1 h, 55%; b) TFA, CH₂Cl₂, rt, 2 h, 100%; c) 4(2-tosylethyl)piperidine, HATU, DIPEA, DMF, rt, 2 h, 61%.
Radiolabeling of these two precursors was then implemented on a C-11 Pro 2 automate (iPhase technologies) for carbon-11 labeling of **46** or on an AllInOne (Trasis) automate for fluorine-18 labeling of **47** (Scheme 6, see details of radiosynthesis optimization in SI, section IV).

Radiofluoration was performed with a starting activity of 32.7 ± 4.5 GBq (EOB) by reaction of precursor **47** with dry K[$^{18}$F]F/K$_{222}$ complex in acetonitrile at 90 °C for 10 min. Longer reaction times or higher temperatures led to degradation of the precursor and lower radiochemical yields. Purification of the crude reaction mixture was then carried out by semi-preparative HPLC on a Symmetry C18 column (eluent: CH$_3$CN/H$_2$O/TFA). Final formulation on a C18 cartridge gave 3.3 ± 0.6 GBq (non-decay corrected) of ready-to-inject [$^{18}$F]**41** in 60 minutes in a 16.6 ± 5.0% decay-corrected radiochemical yield (n = 3). The isolated Identity and purity of the radioligand was confirmed by analytical HPLC with both UV (310 nm) and gamma detection. [$^{18}$F]**41** was obtained with radiochemical and chemical purities above 99% and a decay-corrected molar activity of 51.3 ± 38.3 GBq/µmol (n = 3), compatible with in vivo injections.

Radiomethylation was carried out by reacting 12.2 ± 4.6 GBq of methylating agent [$^{11}$C]CH$_3$OTf and precursor **46** in acetone in basic conditions (1 M sodium hydroxide) at 80°C for 5 minutes. Purification of the crude reaction mixture was then carried out by semi-preparative HPLC on a Symmetry C18 column (eluent: 0.1 M NH$_4$OAc pH 10/acetonitrile). Final formulation on a C18 cartridge gave 935.6 ± 305.1 MBq (non-decay corrected) of ready-to-inject [$^{11}$C]**37** in 45 minutes in a 19.7 ± 3.5% decay-corrected radiochemical yield (n = 14). Identity and purity of the radioligand was confirmed by analytical HPLC with both UV (310 nm) and gamma detection. [$^{11}$C]**37** was obtained with radiochemical and chemical purities above 99% and a decay-corrected molar activity of 40.9 ± 23.3 GBq/µmol (n = 14), compatible with in vivo injections.
<figure><img src="image_12.png" /><figcaption>**Scheme 6.** Radiosynthesis of [¹¹C]37 and [¹⁸F]41</figcaption></figure>

## Autoradiography

**Heterologous displacement of a specific P2Y₁₂R ligand by 37 and 41.** Autoradiography of brain slices from an AAV-*h*P2Y₁₂R rat model, overexpressing the human P2Y₁₂ receptor on one side of the brain (ipsilateral side), was performed with [³H]PSB-22219²³ (see structure in SI, section II). A high unilateral binding, corresponding to the brain region transfected with AAV-*h*P2Y₁₂R, was observed (Figure 4). Decrease of radioactive signal induced by addition of 37 or 41 to the incubation media was quantified by an autoradiography imager. Heterologous displacement with 1 µM of AZD1283, a specific P2Y₁₂R ligand,²⁵ showed that 33 ± 6% of the total binding of [³H]PSB-22219 was non-displaceable (see SI, section II). Displacement of total binding of
[³H]PSB-22219 by 1 µM of **41** (70 ± 5%) was comparable to displacement induced by AZD1283 (67 ± 6%), while compound **37** (1 µM) displaced 35 ± 4% of the total [³H]PSB-22219 signal.

<figure><img src="image_13.png" /></figure>

**Figure 4.** Heterologous displacement of [³H]PSB-22219 on an AAV-*h*P2Y₁₂R rat model. (A) Immunohistochemistry of brain slices of an AAV-*h*P2Y₁₂R rat model; (B) autoradiography images and (C) quantification of displacement of [³H]PSB-22219 by **37** and **41**.

**Homologous displacement of compounds 37 and 41.** Autoradiography with [¹¹C]37 or [¹⁸F]41 was also carried out on brain slices of the AAV-*h*P2Y₁₂R rat model and a non-human primate (NHP) brain.²⁶ In the AAV-*h*P2Y₁₂R-transfected striatum, no increase in binding was observed for either radioligand (Figure 5 - A), even though immunofluorescence data showed strong expression of the human P2Y₁₂ receptor (Figure 5 - C). Nonetheless, displacement studies indicate specific binding to rat brain tissue (Figure 5 - A). Similar results were obtained on NHP brain tissue. Displacement experiments with 10 µM of **41** resulted in 58% and 61% decrease of the total [¹⁸F]41 signal on rat and NHP brain tissue sections, respectively (Figure 5 - B), whereas 10 µM of unlabeled **37** only displaced 24% of the total signal measured with [¹¹C]37 on rat brain tissue sections. The values observed for the homologous displacements of [¹¹C]37 or [¹⁸F]41 by **37** and **41**, respectively, are comparable to the ones obtained in heterologous displacements of a specific P2Y₁₂R ligand with **37** and **41** (Figure 4). These data suggest off-target binding of [¹⁸F]41 and
[¹¹C]37 to another biological target abundantly present in the brain, as has been recently shown by Steén et al for [³H]AZ12464237.²⁷ However, IHC data show abundant microglia expression of endogenous P2Y₁₂ receptor, suggesting widespread binding of [¹⁸F]41 and [¹¹C]37 to the rat P2Y₁₂ receptor throughout the brain (see supplementary data, part II).

<figure><img src="image_14.png" /><figcaption>**Figure 5.** Autoradiography experiments of AAV-hP2Y₁₂R and NHP brain slices with [¹¹C]37 or [¹⁸F]41. (A) Autoradiography images of binding and blocking experiments; (B) Displacement studies with 10 µM reference compound (C) IHC images of AAV-hP2Y₁₂R rat model.</figcaption></figure>

## In vivo evaluation of radioligands

**Biodistribution of [¹⁸F]41 in healthy rats.** The biodistribution of [¹⁸F]41 was evaluated in healthy male SD rats at 5, 15, 30 and 60 minutes p.i. (n = 2 per time point) in blood, plasma, peripheral organs, whole brain, and cerebellum (Figure 6 - A). A low uptake was observed in the brain, with a 0.032%ID/g at 5 minutes p.i., reaching 0.02%ID/g at 60 minutes p.i. This data demonstrated that a low amount of the tracer was able to cross the BBB and the entrance into the
brain was limited. The high uptake observed in the liver followed by a rapid washout and the concomitant increase of the radioactivity in the intestine throughout time suggested a preferential hepatobiliary elimination of the parent [¹⁸F]41 (Figure 6 - A and B). The kidney uptake probably reflected the elimination of [¹⁸F]radiometabolites, more polar than the native tracer, as observed in metabolic profiles in plasma (see in vivo metabolism). The bone uptake was low (0.16% ID/g at 60 min) demonstrating a weak in vivo defluorination, in agreement with the in vitro metabolism study. With the exception of the organs of metabolism and excretion, a low uptake followed by a rapid washout was observed in the other organs such as heart, lung, spleen, pancreas, bone marrow and muscle, indicating a fast distribution of [¹⁸F]41 throughout the body with a rapid washout. Overall, the biodistribution of [¹⁸F]41 demonstrated a weak brain penetration of this radiotracer, hampering its further use as a radiotracer for neuroinflammation (time-activity curves of [¹⁸F]41 can be found in the SI, section V.2).

<figure><img src="image_15.png" /><figcaption>**Figure 6.** Biodistribution of [¹⁸F]41 in healthy rats A- Ex vivo biodistribution of [¹⁸F]41 in blood, plasma and several organs of healthy male SD rats at different time intervals (n = 2/time point). B- Ex vivo biodistribution of [¹⁸F]41 in plasma, brain and cerebellum of healthy male SD rats at different time intervals (n = 2/time point). Data are expressed as percentage of injected dose per g of tissue (mean ± SD).</figcaption></figure>
*In vivo metabolism of [¹⁸F]41.* For in vivo metabolism studies, blood samples were collected 5, 15, 30 and 60 minutes post injection and immediately deproteinated with acetonitrile, centrifuged and the whole supernatants were analyzed by radio-HPLC. Typical HPLC radiochromatograms of plasma and brain extracts are presented in Figure 7 (A, B). Four radiolabeled metabolites [¹⁸F]M1 to [¹⁸F]M4, more polar than the parent tracer were detected in plasma (Figure 7 - C). Parent [¹⁸F]41 accounted for 73% of the recovered activity in plasma samples at 15 minutes *p.i.*, while 15% of the total radioactivity can be addressed to the main highly polar radioactive peak [¹⁸F]M2. The amount of intact radiotracer was further reduced at 60 minutes *p.i.* to 41% with a concomitant increase of [¹⁸F]M2 to 32%. Based on their retention time and in vitro metabolites in RLM, these radiometabolites were likely to be issued from hydroxylations of [¹⁸F]41. The radioactivity measured in the acetonitrile protein precipitates (acetonitrile pellet) remained stable between 5 and 30 minutes (around 5%) and accounted for 8.5% of total plasma radioactivity at 60 min. This low value, which did not significantly increase as a function of time, suggested a very low level of *in vivo* defluorination, in agreement with the *in vitro* metabolism data (0.7% of total metabolites for the proposed defluorinated compound, see SI). The time-course of intact tracer in rat plasma suggested a relatively low metabolism of the tracer. [¹⁸F]M2 and [¹⁸F]M3 were the only radiometabolites detected in the brain (Figure 7 - D). Parent [¹⁸F]41 accounted for 38% of the recovered activity in brain samples at 60 minutes *p.i.*, while 46% of the total radioactivity can be addressed to [¹⁸F]M2.
<figure><img src="image_16.png" /><figcaption>**Figure 7.** In vivo metabolism of [¹⁸F]41 in plasma and brain. Representative radio-HPLC profiles of acetonitrile extracts of rat plasma (A) and rat brain (B) at 60 minutes p.i.; Percentages of parent [¹⁸F]41 and its radiometabolites of healthy SD rats as a function of time p.i. as detected by radio-HPLC in plasma (C) and brain (D).</figcaption></figure>

*In vivo PET imaging of [¹⁸F]41 in rats.*

[¹⁸F]41 was injected intravenously (31.4 ± 14.3 MBq) to healthy SD rats and in the AAV-hP2Y₁₂R rat models and dynamic PET acquisition (60 min) started with radioligand injection. Dynamic brain PET imaging showed low uptake of [¹⁸F]41 (SUVmean 0.11 ± 0.03 and 0.11 ± 0.03) in the left and right striatum, respectively, of healthy or AAV-hP2Y₁₂R injected rats (Figure 8). No significant difference in radioligand binding was found in the transfected versus contralateral striatum (SUVmean 0.13 ± 0.01 and 0.14 ± 0.01 for left and right striatum, respectively; p = 0.2152).
Time-activity curves (TACs) for rat left and right striatum do not differ and look quite similar for normal versus AAV-injected animals (Figure 8 - A & B, blue symbols). Due to the low radioligand uptake in the brain and results from the literature indicating efflux substrate behaviour for $P2Y_{12}$ receptor ligands,²⁰ we performed in vivo PET by blocking the P-glycoprotein (P-gp) and breast cancer resistance efflux transporter using the specific, non-competitive P-gp inhibitor tariquidar.

A significant rise in [¹⁸F]41 uptake was detected under blocking conditions, with an increase in brain uptake that reaches maximum values around 40 to 45 minutes after injection (Figure 8, red symbols). Area under the curve (AUC) values for [¹⁸F]41 increased significantly from 5.7 ± 1.85 to 21.1 ± 6.5, p < 0.0048 in normal rats and 6.7 ± 1.2 to 15.8 ± 4.7, p < 0.0119 in the AAV-hP2Y₁₂R-injected striatum under pre-treatment with tariquidar (Figure 8, A & B, red vs blue), however no significant differences between the ipsilateral and contralateral brain hemispheres could be detected. Similar in vivo experiments were carried out with [¹¹C]37, but no significant differences were found with tariquidar for this radioligand in the normal or AAV-injected rats (see SI). These in vivo PET imaging observations further confirm the inability of [¹¹C]37 to cross the BBB and indicate an efflux substrate behaviour of [¹⁸F]41, which additionally fails to bind to the region of interest, indicating its unsuitability as a brain radiotracer.
<figure><img src="image_17.png" /><figcaption>**Figure 8.** Time Activity Curves and representative PET images of [¹⁸F]41 in healthy rats with (+TQD) and without (-TQD) tariquidar (A) and in AAV-hP2Y₁₂R rats with (+TQD) and without (-TQD) tariquidar (B), n = 3 for all experiments.</figcaption></figure>

*In vivo metabolism and brain kinetics of [¹¹C]37 in non-human primate*

As P-gp expression is lower in non-human primates,²⁸ [¹¹C]37 was injected to NHP in order to evaluate BBB crossing. [¹¹C]37 was injected intravenously to healthy NHPs and showed a moderate metabolism in the arterial blood with a parent fraction of 76% at 15 minutes, 66% at 30 minutes, 43% at 60 minutes, and 40% at 90 minutes after tracer injection (Figure 9 - A). Plasma radioactivity peaked rapidly with a SUVmax of 2.8 and a time of maximum uptake [Tmax] at 1.25 minutes, followed by a rather fast decrease in plasma radioactivity (Figure 9 - B). Regarding brain kinetics, time-activity curves (TACs) in a subset of brain regions showed that [¹¹C]37 entered the brain rapidly, reaching maximal uptake 1.15 minutes after injection, followed by a relatively slow terminal elimination phase. The highest SUV uptake was observed in the temporal cortex, followed by the frontal cortex and the orbitofrontal cortex (Figure 9 - D). The PET data were modeled to
estimate the tissue distribution of [¹¹C]37 in each VOI: the whole brain (V~T~ = 0.51 ml/cm³), frontal cortex (V~T~ = 1.0 ml/cm³), cingulate cortex (V~T~ = 0.3 ml/cm³), temporal cortex (V~T~ = 1.1 ml/cm³), orbitofrontal cortex (V~T~ = 0.7 ml/cm³), parietal cortex (V~T~ = 0.5 ml/cm³), and cerebellum (V~T~ = 0.3 ml/cm³). V~T~ values lower than 1 ml/cm³ in the Logan model are generally indicative of limited penetration of the tracer into the brain, suggesting that the [¹¹C]37 does not efficiently cross the blood-brain barrier. The one or two-compartment modeling was not possible, probably due to a lack of signal intensity at the brain level. We observed comparable low brain uptakes of the radioligand in healthy rats and NHP, preventing further in vivo studies. Similar results have already been observed with piperazine-containing radioligands.²⁹

A

Plasma metabolism

<figure><img src="image_18.png" /></figure>

B

Metabolite-corrected
plasma input function

<figure><img src="image_19.png" /></figure>

C

<figure><img src="image_20.png" /></figure>

D

Average time-activity curves in brain

<figure><img src="image_21.png" /></figure>

**Figure 9.** [¹¹C]37 in vivo metabolism and brain kinetics in a cynomolgus NHP. (A) Blood measures after i.v. [¹¹C]37 injection. Parental fraction (in %) was fitted with a one-exponential
function based on blood data acquired up to 90 minutes after tracer injection. (B) Average
metabolite-corrected plasma input function. (C) Representative 0–90 minutes summed PET co-
registered to T2-weighted MR images. (D) TAC in the whole brain, frontal cortex, cingulate
cortex, temporal cortex, orbitofrontal cortex, parietal cortex, and cerebellum.

CONCLUSION

We report here the design of eight new P2Y12R ligands. Two selected compounds, [18F]41 and [11C]37, were radiolabeled and evaluated in control SD and AAV-hP2Y12R-injected rats. Autoradiography studies using [18F]41 and [11C]37 showed homogeneous, but specific displaceable binding of both radioligands on brain slices obtained from control rats, from AAV-hP2Y12R rat models and from NHP. Nevertheless, an excess of compounds 41 or 37 displaced the P2Y12R ligand [3H]PSB-22219, proving the affinity of compounds 41 or 37 for the human P2Y12R.

Although we cannot exclude off-target binding, we hypothesize that these radioligands also bind to endogenous rodent or endogenous NHP P2Y12R, which might be an explanation for the homogenous signal observed for [11C]37 on AAV-hP2Y12R rat and for [18F]41 on AAV-hP2Y12R rat and NHP brain slices. In vivo, [18F]41 metabolism studies in rats showed high metabolic stability with up to 41% parent radiotracer after 60 minutes, in accordance with the low metabolism observed in in vitro experiments using RLM. In vivo PET imaging experiments with [18F]41 in healthy rats or with [11C]37 in healthy NHP revealed a low uptake into the brain, which is probably due to efflux transport. Efflux transport blocking studies with tariquidar showed a 3- to 4-fold increase of activity in the brain. Future work will focus on refining ligand design to improve BBB permeation and reduce efflux transport by P-glycoprotein.
# EXPERIMENTAL SECTION

## Chemistry

Syntheses of compounds **1-36** and **45** can be found in the supplementary information.

All commercially available reagents and solvents were used without further purification. Purifications of products were carried out by column chromatography using Merck silica gel (63-200 µm). ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) were measured on a Brucker Avance 400 MHz spectrometer. Chemical shifts are reported in parts per million (ppm, δ) downfield from residual solvents peaks and coupling constants are reported in Hertz (Hz). Splitting patterns are designated as singlet (s), broad singlet (br. s), doublet (d), triplet (t), quartet (q) and quintet (quin). Splitting patterns that could not be interpreted or easily visualized are designated as multiplet (m). Melting points (M.p.) were measured on an Electrothermal IA9000 series and are reported in °C. High-resolution mass spectra (HRMS) were performed by the SALSA platform from ICOA laboratory (UMR 7311) on a Bruker maXis mass spectrometer coupled to a Dionex Ultimate 3000 HPLC system (Thermo).

**General protocol for coupling of 33-36 with amines:** HATU (0.16 mmol, 1.6 eq), DIPEA (0.16 mmol, 1.6 eq) and appropriate amine (1.6 mmol, 1.6 eq) were added to a solution of **33-36** (0.10 mmol 1 eq) in DMF (1 mL) and the solution was stirred for 1 h at room temperature. The reaction mixture was then diluted with EtOAc and washed with a 5% LiCl aqueous solution (3 x). The organic layer was dried over sodium sulfate, filtered and concentrated *in vacuo*. The crude product was purified by silica gel chromatography.

**N-[6-(4-Butanoyl-5-methyl-1H-pyrazol-1-yl)pyridazin-3-yl]-5-chloro-1-[2-(4-methylpiperazin-1-yl)-2-oxoethyl]-1H-indole-3-carboxamide 37.** Prepared following general procedure starting from **33**. Expected product **37** was obtained as a white solid in 48% yield
(purification: silica gel, DCM/MeOH 95/5 to 90/10). ¹H-NMR (DMSO-d₆, 400 MHz): 11.36 (s, 1H, NH), 8.72 (d, 1H, *J* = 9.3 Hz, *H*<sub>ar</sub>), 8.62 (s, 1H, *H*<sub>ar</sub>), 8.42 (s, 1H, *H*<sub>ar</sub>), 8.23 (d, 1H, *J* = 2.1 Hz, *H*<sub>ar</sub>), 8.13 (d, 1H, *J* = 9.3 Hz, *H*<sub>ar</sub>), 7.56 (d, 1H, *J* = 8.2 Hz, *H*<sub>ar</sub>), 7.30 (dd, 1H, *J* = 8.2 Hz and 2.1 Hz, *H*<sub>ar</sub>), 5.37 (s, 2H, *CH*₂), 3.62-3.58 (m, 2H, *CH*₂ piperazine), 3.51-3.46 (m, 2H, *CH*₂ piperazine), 2.87 (t, 2H, *J* = 7.6 Hz, *CH*₂), 2.82 (s, 3H, *CH*₃), 2.46-2.42 (m, 2H, *CH*₂ piperazine), 2.34-2.30 (m, 2H, *CH*₂ piperazine), 2.24 (s, 3H, *NCH*₃), 1.65 (sext, 2H, *J* = 7.6 Hz, *CH*₂), 0.95 (t, 3H, *J* = 7.6 Hz, *CH*₃). ¹³C-NMR (DMSO-d₆, 100 MHz): 195.7 (C=O), 165.0 (C=O), 163.2 (C=O), 155.8 (C<sub>ar</sub>), 152.4 (C<sub>ar</sub>), 143.5 (CH<sub>ar</sub>), 142.8 (C<sub>ar</sub>), 136.4 (CH<sub>ar</sub>), 135.8 (C<sub>ar</sub>), 127.8 (C<sub>ar</sub>), 126.2 (C<sub>ar</sub>), 124.7 (CH<sub>ar</sub>), 122.5 (CH<sub>ar</sub>), 122.0 (CH<sub>ar</sub>), 121.3 (CH<sub>ar</sub>), 120.2 (C<sub>ar</sub>), 112.7 (CH<sub>ar</sub>), 108.2 (C<sub>ar</sub>), 54.6 (CH₂ piperazine), 54.2 (CH₂ piperazine), 47.8 (NCH₂), 45.6 (CH₃), 44.2 (CH₂ piperazine), 42.3 (CH₂), 41.5 (CH₂ piperazine), 17.2 (CH₂), 13.6 (CH₃), 12.5 (CH₃). M.p.: 164-165 °C. LC-MS (ESI): m/z calculated for C₂₈H₃₂ClN₈O₃ [M+H]⁺: 563.2, 565.2; found: 563.2, 565.2.

*N*--[6-(4-Butanoyl-5-methyl-1H-pyrazol-1-yl)pyridazin-3-yl]-5-methyl-1-[2-(4-methylpiperazin-1-yl)-2-oxoethyl]-1H-indole-3-carboxamide 38. Prepared following general procedure starting from 34. Expected product 38 was obtained as a white solid in 43% yield (purification: silica gel, DCM/MeOH 95/5 to 90/10). ¹H-NMR (DMSO-d₆, 400 MHz): 11.19 (s, 1H, NH), 8.72 (d, 1H, *J* = 9.8 Hz, *H*<sub>ar</sub>), 8.51 (s, 1H, *H*<sub>ar</sub>), 8.41 (s, 1H, *H*<sub>ar</sub>), 8.11 (d, 1H, *J* = 9.8 Hz, *H*<sub>ar</sub>), 8.05 (m, 1H, *H*<sub>ar</sub>), 7.37 (d, 1H, *J* = 8.2 Hz, *H*<sub>ar</sub>), 7.08 (dd, 1H, *J* = 8.2, *J* = 1.5 Hz, *H*<sub>ar</sub>), 5.28 (s, 2H, *CH*₂), 3.62-3.59 (t, 2H, *J* = 4.8 Hz, *CH*₂ piperazine), 3.50-3.47 (t, 2H, *J* = 4.8 Hz, *CH*₂ piperazine), 2.87 (t, 2H, *J* = 7.2 Hz, *CH*₂), 2.82 (s, 3H, *CH*₃), 2.45-2.41 (m, 5H, *CH*₂ piperazine and *CH*₃) 2.33-2.29 (m, 2H, *CH*₂ piperazine), 2.23 (s, 3H, *NCH*₃) 1.65 (sext, 2H, *J* = 7.2 Hz, *CH*₂), 0.95 (t, 3H, *J* = 7.2 Hz, *CH*₃). ¹³C-NMR (DMSO-d₆, 100 MHz): 195.7 (C=O), 165.2 (C=O), 163.6
(C=O), 156.0 (Car), 152.3 (Car), 143.5 (CHar), 142.8 (Car), 135.6 (CHar), 135.1 (Car), 130.2 (Car), 127.0 (Car), 124.6 (CHar), 123.9 (CHar), 121.8 (CHar), 121.3 (Car), 120.8 (CHar), 110.5 (CHar), 108.1 (Car), 54.6 (CH2 piperazine), 54.2 (CH2 piperazine), 47.6 (CH2), 45.6 (CH3), 44.2 (CH2 piperazine), 42.3 (CH2), 41.5 (CH2 piperazine), 21.3 (CH3), 17.3 (CH2), 13.6 (CH3), 12.5 (CH3). M.p.: 189-190 °C. LC-MS (ESI): m/z calculated for C29H35N8O3 [M+H]⁺: 543.28; found 543.25.

**N-(4-(4-Butyryl-5-methyl-1H-pyrazol-1-yl)phenyl)-5-chloro-1-(2-(4-methylpiperazin-1-yl)-2-oxoethyl)-1H-indole-3-carboxamide 39.** Prepared following general procedure starting from **35**. Expected product **39** was obtained as a pale yellow powder in 84% yield (purification: silica gel, DCM/MeOH 95/5 to 90/10). **1H-NMR (DMSO-d6, 400 MHz):** 10.11 (s, 1H, NH), 8.31 (s, 1H, Har), 8.24 (s, 1H, Har), 8.21 (d, 1H, *J* = 2.1 Hz, Har), 7.95 (d, 2H, *J* = 8.8 Hz, 2 Har), 7.54 (d, 1H, *J* = 8.7 Hz, Har), 7.49 (d, 2H, *J* = 8.8 Hz, Har), 7.26 (dd, 1H, *J* = 8.7, *J* = 2.1 Hz, Har), 5.37 (s, 2H, NCH2), 3.60-3.57 (m, 2H, CH2 piperazine), 3.48-3.46 (m, 2H, CH2 piperazine), 2.81 (t, 2H, *J* = 7.1 Hz, CH2), 2.52 (s, 3H, CH3), 2.45-2.42 (m, 2H, CH2 piperazine), 2.31-2.28 (m, 2H, CH2 piperazine), 2.23 (s, 3H, NCH3), 1.63 (sext, 2H, *J* = 7.2 Hz, CH2), 0.94 (t, 3H, *J* = 7.1 Hz, CH3). **13C-NMR (DMSO-d6, 100 MHz):** 195.5 (C=O), 165.2 (C=O), 162.6 (C=O), 142.1 (Car), 141.4 (Car), 139.9 (Car), 135.8 (Car), 134.7 (Car), 132.9 (Car), 127.7 (Car), 125.8 (Car), 125.7 (2 Car), 124.0 (Car), 122.2 (Car), 120.3 (Car), 119.8 (2 Car), 112.4 (Car), 109.8 (Car), 54.5 (CH2), 54.1 (CH2), 47.6 (NCH2), 45.5 (CH3), 44.0 (CH2), 42.0 (CH2), 41.4 (CH2), 17.4 (CH2), 13.7 (CH3), 11.9 (CH3). M.p.: 82-83 °C. **HRMS (ESI):** m/z calculated for C30H34ClN6O3 [M+H]⁺: 561.2375; found: 561.2366.

**N-(4-(4-Butyryl-5-methyl-1H-pyrazol-1-yl)phenyl)-5-methyl-1-(2-(4-methylpiperazin-1-yl)-2-oxoethyl)-1H-indole-3-carboxamide 40.** Prepared following general procedure starting from **36**. Expected product **40** was obtained as a white solid in 69% yield (purification: silica gel, DCM/MeOH 95/5 to 80/20). **1H-NMR (DMSO-d6, 400 MHz):** 10.00 (s, 1H, NH), 8.24 (s, 1H,
$H_{\text{ar}}$), 8.19 (s, 1H, $H_{\text{ar}}$), 8.02 (m, 1H, $H_{\text{ar}}$), 7.96 (d, 2H, $J = 9.1 \text{ Hz, } 2 H_{\text{ar}}$), 7.47 (d, 2H, $J = 9.1 \text{ Hz, } 2 H_{\text{ar}}$), 7.35 (d, 1H, $J = 8.5 \text{ Hz, } H_{\text{ar}}$), 7.05 (dd, 1H, $J = 8.5, J = 1.6 \text{ Hz, } H_{\text{ar}}$), 5.30 (s, 2H, $NCH_2$), 3.61-3.57 (m, 2H, $CH_2$ piperazine), 3.49-3.46 (m, 2H, $CH_2$ piperazine), 2.81 (t, 2H, $J = 7.2 \text{ Hz, } CH_2$), 2.52 (s, 3H, $CH_3$), 2.44-2.40 (m, 5H, $CH_2$ piperazine and $CH_3$), 2.31-2.27 (m, 2H, $CH_2$ piperazine), 2.22 (s, 3H, $CH_3$), 1.64 (sext, 2H, $J = 7.2 \text{ Hz, } CH_2$), 0.94 (t, 3H, $J = 7.2, CH_3$). ¹³C-NMR (DMSO-d₆, 100 MHz): 195.5 (C=O), 165.4 (C=O), 163.1 (C=O), 142.1 ($C_{\text{ar}}$), 141.5 ($C_{\text{ar}}$), 140.2 ($C_{\text{ar}}$), 135.6 ($C_{\text{ar}}$), 133.4 ($C_{\text{ar}}$), 132.6 ($C_{\text{ar}}$), 129.7 ($C_{\text{ar}}$), 126.8 ($C_{\text{ar}}$), 125.7 (2 $C_{\text{ar}}$), 123.7 ($C_{\text{ar}}$), 120.8 ($C_{\text{ar}}$), 120.2 ($C_{\text{ar}}$), 119.6 (2 $C_{\text{ar}}$), 110.3 ($C_{\text{ar}}$), 109.2 ($C_{\text{ar}}$), 54.6 ($CH_2$), 54.2 ($CH_2$), 47.3 ($CH_2$), 45.7 ($CH_3$), 44.2 ($CH_2$), 41.9 ($CH_2$), 41.5 ($CH_2$), 21.3 ($CH_3$), 17.4 ($CH_2$), 13.7 ($CH_3$), 11.9 ($CH_3$).

**M.p.:** 153-154 °C. **HRMS (ESI):** $m/z$ calcd for $C_{31}H_{37}N_6O_3 [M+H]⁺$: 541.2922; found: 541.2922.

*N-(4-(4-Butyryl-5-methyl-1H-pyrazol-1-yl)phenyl)-5-chloro-1-(2-(4-(2-fluoroethyl)piperidin-1-yl)-2-oxoethyl)-1H-indole-3-carboxamide 41.* Prepared following general procedure starting from 35. Expected product 41 was obtained as a white solid in 75% yield (purification: silica gel, DCM/MeOH 99/1 to 93/7). ¹H-NMR (DMSO-d₆, 400 MHz): 10.12 (s, 1H, NH), 8.31 (s, 1H, $H_{\text{ar}}$), 8.25 (s, 1H, $H_{\text{ar}}$), 8.21 (d, 1H, $J = 2.0 \text{ Hz, } H_{\text{ar}}$), 7.95 (d, 2H, $J = 8.9 \text{ Hz, } 2 H_{\text{ar}}$), 7.53 (d, 1H, $J = 8.8 \text{ Hz, } H_{\text{ar}}$), 7.49 (d, 2H, $J = 8.9 \text{ Hz, } 2 H_{\text{ar}}$), 7.26 (dd, 1H, $J = 8.8, J = 2.0 \text{ Hz, } H_{\text{ar}}$), 5.40 (d, 1H, $J = 17.4 \text{ Hz, CHH-N}$), 5.33 (d, 1H, $J = 17.4 \text{ Hz, CHH-N}$), 4.54 (dt, 2H, $J = 47.5 \text{ Hz, } J = 5.9 \text{ Hz, } CH_2F$), 4.31 (m, 1H, CH piperidine), 3.98 (m, 1H, CH piperidine), 3.14 (t, 1H, $J = 13.9 \text{ Hz, CH piperidine}$), 2.81 (t, 2H, $J = 7.2 \text{ Hz, } CH_2$), 2.63 (m, 1H, CH piperidine), 2.52 (s, 3H, $CH_3$), 1.82 (m, 1H, CH piperidine), 1.75-1.59 (m, 5H, 2 $CH_2$, CH piperidine), 1.33-1.23 (m, 2H, 2 CH piperidine), 1.04 (m, 1H, CH piperidine), 0.94 (t, 3H, $J = 7.2 \text{ Hz, } CH_3$). ¹³C-NMR (DMSO-d₆, 100 MHz): 195.5 (C=O), 164.8 (C=O), 162.6 (C=O), 142.1 ($C_{\text{ar}}$), 141.5 ($C_{\text{ar}}$), 139.9 ($C_{\text{ar}}$), 135.8 ($C_{\text{ar}}$), 134.7 ($C_{\text{ar}}$), 132.8 ($C_{\text{ar}}$), 127.7 ($C_{\text{ar}}$), 125.8 ($C_{\text{ar}}$), 125.7 (2 $C_{\text{ar}}$), 122.2 ($C_{\text{ar}}$),
120.2 (2 C<sub>ar</sub>), 119.8 (2 C<sub>ar</sub>), 112.5 (C<sub>ar</sub>), 109.3 (C<sub>ar</sub>), 81.9 (d, J = 161.3 Hz, CH<sub>2</sub>F), 47.6 (CH<sub>2</sub>), 44.4 (C piperidine), 41.9 (CH<sub>2</sub>), 41.8 (C piperidine), 36.2 (d, J = 18.5 Hz, CH<sub>2</sub>CH<sub>2</sub>F), 32.0 (C piperidine), 31.9 (C piperidine), 31.3 (C piperidine), 17.4 (CH<sub>2</sub>), 13.7 (CH<sub>3</sub>), 11.9 (CH<sub>3</sub>). M.p.: 185-186 °C. HRMS (ESI): m/z calcd for C<sub>32</sub>H<sub>36</sub>ClFN<sub>5</sub>O<sub>3</sub> [M+H]<sup>+</sup>: 592.2485; found: 592.2481.

*N-(4-(4-Butyryl-5-methyl-1H-pyrazol-1-yl)phenyl)-1-(2-(4-(2-fluoroethyl)piperidin-1-yl)-2-oxoethyl)-5-methyl-1H-indole-3-carboxamide* **42**. Prepared following general procedure starting from **36**. Expected product **42** was obtained as a white solid in 43% yield (purification: silica gel, DCM/MeOH 98/2 to 90/10). **1H-NMR** (DMSO-d<sub>6</sub>, 400 MHz): 9.98 (s, 1H, NH), 8.23 (s, 1H, H<sub>ar</sub>), 8.18 (s, 1H, H<sub>ar</sub>), 8.03 (s, 1H, H<sub>ar</sub>), 7.96 (d, 2H, J = 8.8 Hz, 2 H<sub>ar</sub>), 7.47 (d, 2H, J = 8.8 Hz, 2 H<sub>ar</sub>), 7.35 (d, 1H, J = 8.5 Hz, H<sub>ar</sub>), 7.05 (d, 1H, J = 8.5 Hz, H<sub>ar</sub>), 5.30 (d, 1H, J = 17.0 Hz, CHH-N), 5.24 (d, 1H, J = 17.0 Hz, CHH-N), 4.54 (dt, 2H, J = 47.1 Hz, J = 5.6 Hz, CH<sub>2</sub>F), 4.32 (m, 1H, CH piperidine), 4.01 (m, 1H, CH piperidine), 3.13 (t, 1H, J = 12.8 Hz, CH piperidine), 2.81 (t, 2H, J = 7.2 Hz, CH<sub>2</sub>), 2.64 (t, 1H, J = 12.2 Hz, CH piperidine), 2.52 (s, 3H, CH<sub>3</sub>), 2.43 (s, 3H, CH<sub>3</sub>), 1.82-1.60 (m, 6H, 3 CH<sub>2</sub>), 1.31-1.22 (m, 2H, 2 CH piperidine), 1.05 (m, 1H, CH piperidine), 0.94 (t, 3H, J = 7.2 Hz, CH<sub>3</sub>). **13C-NMR** (DMSO-d<sub>6</sub>, 100MHz): 195.5 (C=O), 165.0 (C=O), 163.1 (C=O), 142.1 (C<sub>ar</sub>), 141.4 (C<sub>ar</sub>), 140.2 (C<sub>ar</sub>), 135.6 (C<sub>ar</sub>), 133.3 (C<sub>ar</sub>), 132.6 (C<sub>ar</sub>), 129.7 (C<sub>ar</sub>), 126.8 (C<sub>ar</sub>), 125.7 (2 C<sub>ar</sub>), 123.7 (C<sub>ar</sub>), 120.8 (C<sub>ar</sub>), 120.2 (C<sub>ar</sub>), 119.7 (2 C<sub>ar</sub>), 110.3 (C<sub>ar</sub>), 109.2 (C<sub>ar</sub>), 81.8 (d, J = 161.5 Hz, CH<sub>2</sub>F), 47.4 (CH<sub>2</sub>), 44.4 (CH<sub>2</sub>), 42.0 (CH<sub>2</sub>), 41.7 (CH<sub>2</sub>), 36.2 (d, J = 19.0 Hz, CH<sub>2</sub>), 32.0 (CH<sub>2</sub>), 31.9 (CH), 31.3 (CH<sub>2</sub>), 21.2 (CH<sub>3</sub>), 17.4 (CH<sub>2</sub>), 13.7 (CH<sub>3</sub>), 11.9 (CH<sub>3</sub>). M.p.: 179-180 °C. HRMS (ESI): m/z calcd for C<sub>33</sub>H<sub>39</sub>FN<sub>5</sub>O<sub>3</sub> [M+H]<sup>+</sup>: 572.3031; found: 572.3029.

*N-(4-(4-Butyryl-5-methyl-1H-pyrazol-1-yl)phenyl)-1-(2-((2-fluoroethyl)amino)-2-oxoethyl)-5-methyl-1H-indole-3-carboxamide* **43**. Prepared following general procedure
starting from **36**. Expected product **43** was obtained as a white solid in 63% yield (purification: silica gel, DCM/MeOH 95/5). **¹H-NMR** (DMSO-d₆, 400 MHz): 10.02 (s, 1H, NH), 8.62 (t, 1H, *J* = 5.5 Hz, NH), 8.26 (s, 1H, *H*ar), 8.24 (s, 1H, *H*ar), 8.04 (s, 1H, *H*ar), 7.97 (d, 2H, *J* = 8.8 Hz, 2 *H*ar), 7.48 (d, 2H, *J* = 8.8 Hz, 2 *H*ar), 7.35 (d, 1H, *J* = 8.3 Hz, *H*ar), 7.08 (dd, 1H, *J* = 8.3, *J* =1.3 Hz, *H*ar), 4.95 (s, 2H, CH₂), 4.47 (dt, 2H, *J* = 47.9 Hz, *J* = 5.1 Hz, F-CH₂), 3.44 (dq, 2H, *J* = 26.7 Hz, *J* = 5.1 Hz, F-CH₂CH₂), 2.81 (t, 2H, *J* = 7.4 Hz, CH₂), 2.52 (s, 3H, CH₃), 2.43 (s, 3H, CH₃), 1.64 (sext, 2H, *J* = 7.4 Hz, CH₂), 0.94 (t, 3H, *J* = 7.4 Hz, CH₃). **¹³C-NMR** (DMSO-d₆, 100MHz): 195.5 (C=O), 167.2 (C=O), 163.1 (C=O), 142.1 (Cₐr), 141.4 (Cₐr), 140.2 (Cₐr), 135.2 (Cₐr), 133.1 (Cₐr), 132.7 (Cₐr), 129.9 (Cₐr), 127.0 (Cₐr), 125.7 (2 Cₐr), 123.9 (Cₐr), 121.0 (Cₐr), 120.2 (Cₐr), 119.7 (2 Cₐr), 110.0 (Cₐr), 109.3 (Cₐr), 82.2 (d, *J* = 166 Hz, FCH₂), 48.9 (CH₂), 42.0 (CH₂), 21.2 (CH₃), 17.4 (CH₂), 13.7 (CH₃), 11.9 (CH₃), (CH₂CH₂F under DMSO-d₆ peak). **M.p.:** 253-254 °C. **HRMS** (**ESI**): m/z calcd for C₂₈H₃₁FN₅O₃ [M+H]⁺: 504.2405; found: 504.2404.

**N-(4-(4-Butyryl-5-methyl-1H-pyrazol-1-yl)phenyl)-1-(2-((2-(2-(2-fluoropropoxy)ethoxy)ethyl)amino)-2-oxoethyl)-5-methyl-1H-indole-3-carboxamide** **44.**

Prepared following general procedure starting from **36**. Expected product **44** was obtained as a white solid in 35% yield (purification: silica gel, DCM/MeOH 98/2 to 93/7). **¹H-NMR** (DMSO-d₆, 400 MHz): 10.05 (s, 1H, NH), 8.47 (t, 1H, *J* = 5.5 Hz, NH), 8.28 (s, 1H, *H*ar), 8.24 (s, 1H, *H*ar), 8.04 (m, 1H, *H*ar), 7.97 (d, 2H, *J* = 8.8 Hz, 2 *H*ar), 7.48 (d, 2H, *J* = 8.8 Hz, 2 *H*ar), 7.35 (d, 1H, *J* = 8.5 Hz, *H*ar), 7.07 (dd, 1H, *J* = 8.5, *J* = 1.5 Hz, *H*ar), 4.92 (s, 2H, CH₂), 4.52 (dt, 2H, *J* = 47.9 Hz, *J* = 4.1 Hz, CH₂F), 3.65 (dt, 2H, *J* = 31.3 Hz, *J* = 4.1 Hz, CH₂CH₂F), 3.58-3.53 (m, 4H, 2 CH₂), 3.46 (t, 2H, *J* = 5.5 Hz, CH₂), 3.28 (q, 2H, *J* = 5.5 Hz, CH₂NH), 2.81 (t, 2H, *J* = 7.3 Hz, CH₂), 2.52 (s, 3H, CH₃), 2.42 (s, 3H, CH₃), 1.63 (sext, 2H, *J* = 7.3 Hz, CH₂), 0.93 (t, 3H, *J* = 7.3 Hz, CH₃). **¹³C-NMR** (DMSO-d₆, 400 MHz): 195.5 (C=O), 166.8 (C=O), 163.1 (C=O), 142.1 (Cₐr), 141.5
(C<sub>ar</sub>), 140.2 (C<sub>ar</sub>), 135.2 (C<sub>ar</sub>), 133.1 (C<sub>ar</sub>), 132.6 (C<sub>ar</sub>), 129.9 (C<sub>ar</sub>), 127.0 (C<sub>ar</sub>), 125.7 (2 C<sub>ar</sub>), 123.9 (C<sub>ar</sub>), 121.0 (C<sub>ar</sub>), 120.2 (C<sub>ar</sub>), 119.7 (2 C<sub>ar</sub>), 110.0 (C<sub>ar</sub>), 109.2 (C<sub>ar</sub>), 83.0 (d, J = 166.0 Hz, CH<sub>2</sub>F), 69.7 (CH<sub>2</sub>), 69.7 (d, J = 18.5 Hz, CH<sub>2</sub>), 69.6 (CH<sub>2</sub>), 68.9 (CH<sub>2</sub>), 49.0 (CH<sub>2</sub>), 41.9 (CH<sub>2</sub>), 21.2 (CH<sub>3</sub>), 17.4 (CH<sub>2</sub>), 13.7 (CH<sub>3</sub>), 11.9 (CH<sub>3</sub>), (CH<sub>2</sub> under DMSO-d6 peak). **M.p.:** 162-163 °C.
**HRMS (ESI):** *m/z* calcd for C<sub>32</sub>H<sub>39</sub>FN<sub>5</sub>O<sub>5</sub> [M+H]<sup>+</sup>: 592.2930; found: 592.2926.

**2-(1-(2-(3-((4-(4-Butyryl-5-methyl-1H-pyrazol-1-yl)phenyl)carbamoyl)-5-chloro-1H-indol-1-yl)acetyl)piperidin-4-yl)ethyl 4-methylbenzenesulfonate 47.** Prepared following general procedure starting from **35**. Expected product **47** was obtained as a white solid in 61% yield (purification: silica gel, DCM/MeOH 99/1 to 93/7). **¹H-NMR (DMSO-d₆, 400 MHz):** 10.10 (s, 1H, NH), 8.28 (s, 1H, H<sub>ar</sub>), 8.24 (s, 1H, H<sub>ar</sub>), 8.20 (d, 1H, J = 2.0 Hz, H<sub>ar</sub>), 7.95 (d, 2H, J = 8.9 Hz, 2 H<sub>ar</sub>), 7.82 (d, 2H, J = 8.9 Hz, 2 H<sub>ar</sub>), 7.52-7.47 (m, 5H, 5 H<sub>ar</sub>), 7.25 (dd, 1H, J = 8.8, J = 2.0 Hz, H<sub>ar</sub>), 5.36 (d, 1H, J = 17.4 Hz, CHH-N), 5.30 (d, 1H, J = 17.4 Hz, CHH-N), 4.24 (m, 1H, CH piperidine), 4.09 (t, 2H, J = 5.8 Hz, CH<sub>2</sub>), 3.92 (m, 1H, CH piperidine), 3.05 (t, 1H, J = 13.9 Hz, CH piperidine), 2.81 (t, 2H, J = 7.2 Hz, CH<sub>2</sub>), 2.54 (m, 1H, CH piperidine), 2.52 (s, 3H, CH<sub>3</sub>), 2.43 (s, 3H, CH<sub>3</sub>), 1.63 (sext, 2H, J = 7.3 Hz, CH<sub>2</sub>), 1.61-1.50 (m, 5H, CH<sub>2</sub>, 3 CH piperidine), 1.26-1.16 (m, 2H, 2 CH piperidine), 0.94 (t, 3H, J = 7.2 Hz, CH<sub>3</sub>). **¹³C-NMR (DMSO-d₆, 100 MHz):** 195.5 (C=O), 164.8 (C=O), 162.6 (C=O), 144.9 (C<sub>ar</sub>), 142.1 (C<sub>ar</sub>), 141.5 (C<sub>ar</sub>), 139.9 (C<sub>ar</sub>), 135.8 (C<sub>ar</sub>), 134.7 (C<sub>ar</sub>), 132.8 (C<sub>ar</sub>), 132.4 (C<sub>ar</sub>), 130.2 (2 C<sub>ar</sub>), 127.7 (C<sub>ar</sub>), 127.6 (2 C<sub>ar</sub>), 125.8 (C<sub>ar</sub>), 125.7 (2 C<sub>ar</sub>), 122.2 (C<sub>ar</sub>), 120.2 (2 C<sub>ar</sub>), 119.8 (2 C<sub>ar</sub>), 112.6 (C<sub>ar</sub>), 109.3 (C<sub>ar</sub>), 68.6 (CH<sub>2</sub> OTs), 47.6 (CH<sub>2</sub>), 44.3 (C piperidine), 41.9 (CH<sub>2</sub>), 41.6 (C piperidine), 34.5 (CH<sub>2</sub>), 31.7 (C piperidine), 31.5 (C piperidine), 30.9 (C piperidine), 21.1 (CH<sub>3</sub>), 17.4 (CH<sub>2</sub>), 13.7 (CH<sub>3</sub>), 11.9 (CH<sub>3</sub>). **M.p.:** 149-150 °C. **HRMS (ESI):** *m/z* calcd for C<sub>39</sub>H<sub>43</sub>ClN<sub>5</sub>O<sub>6</sub>S [M+H]<sup>+</sup>: 744.2617; found: 744.2607.
N-[6-(4-Butanoyl-5-methyl-1H-pyrazol-1-yl)pyridazin-3-yl]-5-chloro-1-[2-(piperazin-1-yl)-2-oxoethyl]-1H-indole-3-carboxamide 46. TFA (100 µL) was added to a solution of 45 (15 mg, 0.024 mmol) in DCM (1 mL). The mixture was stirred for 2 h at room temperature. The reaction mixture was then concentrated under vacuum and product 46 was used without further purification (yellow solid, 13 mg, quant). ¹H NMR (DMSO-d₆, 400 MHz): 11.40 (s, 1H, NH), 9.02 (s, 1H, NH₂ piperazine), 8.71 (d, 1H, *J* = 9.4 Hz, *H*ₐr), 8.57 (s, 1H, *H*ₐr), 8.41 (s, 1H, *H*ₐr), 8.23 (d, 1H, *J* = 2.1 Hz, *H*ₐr), 8.13 (d, 1H, *J* = 9.4 Hz, *H*ₐr), 7.59 (d, 1H, *J* = 9.8 Hz, *H*ₐr), 7.30 (dd, 1H, *J* = 9.8 Hz and 2.1 Hz, *H*ₐr), 5.40 (s, 2H, NCH₂), 3.83-3.79 (m, 2H, CH₂ piperazine), 3.70-3.66 (m, 2H, CH₂ piperazine), 3.31-3.24 (m, 2H, CH₂ piperazine), 3.16-3.12 (m, 2H, CH₂ piperazine), 2.86 (t, 2H, *J* = 7.3 Hz, CH₂), 2.81 (s, 3H,CH₃), 1.64 (sext, 2H, *J* = 7.3 Hz, CH₂), 0.94 (t, 3H, *J* = 7.3 Hz, CH₃). ¹³C NMR (DMSO-d₆, 100 MHz): 195.7 (C=O), 165.3 (C=O), 163.2 (C=O), 158.4 (Cₐr), 155.8 (Cₐr), 152.5 (Cₐr), 143.5 (CHₐr), 142.9 (Cₐr), 136.2 (CHₐr), 135.8 (Cₐr), 127.8 (Cₐr), 126.3 (Cₐr), 124.7 (CHₐr), 122.5 (CHₐr), 122.0 (CHₐr), 121.3 (Cₐr), 120.2 (CHₐr), 108.4 (Cₐr), 47.8 (CH₂), 42.7 (CH₂ piperazine), 42.6 (CH₂ piperazine), 42.3 (CH₂), 41.4 (CH₂ piperazine), 38.5 (CH₂ piperazine), 17.3 (CH₂), 13.6 (CH₃), 12.5 (CH₃). M.p.: 159-160 °C. HRMS (ESI): m/z calcd for C₂₇H₃₀ClN₈O₃ [M+H]⁺: 549.2124; found: 549.2127.

### Radioligand binding assay

The experiments were carried out in a 50 mM Tris-HCl buffer at pH 7.4 in a final volume of 500 µL, which included 5 µL of the tested compound (prepared by serial dilutions), 100 µL of [³H]PSB-22219 at a final concentration of 2 nM, and 100 µL of membrane preparations of Chinese hamster ovary (CHO) cells recombinantly expressing the human P2Y₁₂R in buffer (containing 5 µg of protein, see SI). To assess nonspecific binding, 1 µM of the potent P2Y₁₂R antagonist PSB-0739³⁰ was added. Incubation was conducted for 1 hour at 18 °C, initiated by the addition of the membrane
preparation. Bound radioligand was separated from unbound ligand by filtration through GF/B
glass fiber filters using a Brandel cell harvester (Model M-48, Brandel, MD, USA). Each sample
was washed three times with 3 mL of ice-cold Tris-HCl buffer (50 mM) containing 0.1% Tween
20 at pH 7.4. After washing, the filters were punched out and placed into scintillation vials, where
they were incubated with 2.5 mL of scintillation cocktail (ProSafe FC+, Triskem) for 9 hours. The
radioactivity of the samples was then measured using a Hidex 600 SL Automatic TDCR Liquid
Scintillation Counter, with a counting efficiency of 53-55%. Each experiment was independently
repeated at least three times, with each trial performed in duplicate. pIC50 and pKı values were
calculated using the one-site homologous or one-site fit Kı models as defined in GraphPad Prism
software version 8.4.2 (GraphPad Software Inc., San Diego, CA, USA). The radioligand and its
characterization is described in a recent publication.²³

In vitro metabolic assays

**Chemicals** D-glucose-6-phosphate disodium salt, β-nicotinamide adenine dinucleotide phosphate (NADP+), glucose-6-phosphate dehydrogenase (G6PDH) from yeast, formic acid, trifluoroacetic acid were purchased from Sigma-Aldrich (France). Analytical LC/MS grade solvents were used for UPLC/MS analyses (Thermo-Fisher Scientific, France).

Rat liver microsomes (RLM) and pooled human liver microsomes (HLM) that were fully
characterized for their protein content, P450s concentration and catalytic activities were obtained
from Gibco (Fisher Scientific, France) and stored at -80 °C prior to use. Stock solutions (10 mM)
of P2Y12R ligands synthetized in this study (37-44) were prepared in DMSO. The incubation
mixtures containing 1 mg of microsomal proteins (HLM and MLM), a NADPH-generating system
(5 mM G6P, 1 mM NADP+, 2 UI/assay G6PDH), the P2Y12R ligand at a final concentration of
100 µM and 0.1 M aqueous potassium phosphate buffer, pH 7.4 in a final volume of 1 mL, were
pre-incubated for 2 minutes at 37 °C. The reaction was started by the addition of the NADPH-generating system (200 µL) and was conducted in a 37 °C water bath with gentle shaking for 60 minutes. The reaction was stopped by the addition of an equal volume of acetonitrile (1 mL). The mixtures were centrifuged at 4 °C for 5 minutes at 10,000 g. The resulting supernatants were filtered (Vecta Spin Micro 0.2 mm; Whatman), and analyzed by LC/MS as described below. In control assays, the mixtures were incubated without the NADPH-generating system or without microsomes to confirm the stability of the ligand in the experimental conditions. The percentage of each metabolite was determined as a percentage of the total peak area (UV detection).

The main metabolites of the P2Y₁₂R ligands generated in rat and human liver microsomes were identified by LC/MS and MSn analyses. The main oxidative metabolites generated upon P450 oxidation in microsomal incubates were characterized by LC/MS in the positive mode using an Ion Trap LTQ VelosPro ion mass spectrometer equipped with an electrospray source (Thermo Fisher Scientific, France). The Ultimate 3000 UPLC system interfaced with the LTQ consisted of LC pumps (HPG3400RS series N°8041829), PDA (DAD3000 Series N°8041519) and autosampler (WPS3000TRS Series N°8043592). Pressurized nitrogen was used as sheath gas with a flow rate of 25 units (arbitrary units for sheath gas pressure as defined by the manufacturer). The source voltage for electrospray ionization was 4 kV; source temperature was 250°C; capillary voltage was 40 V and capillary temperature was 350 °C. The parent ligands and their metabolites were separated on a reverse phase UPLC Atlantis C18, 1.8 µm, 2.1x100 mm column (Waters, France). The mobile phase consisted of A: water containing 0.05% formic acid and B: acetonitrile containing 0.05% formic acid. A linear gradient from 20 to 100% of B in 17 minutes was applied to the column at a flow rate of 300 µL/min. The full scan MS acquisition mode was set up to detect ions in the range of *m/z* 50-700. The MS was set up in the MS/MS mode for acquiring, isolating
with a mass width of 1-3 amu (Cl) and dissociating the ions m/z = 561/563 (39), m/z = 504 (43), m/z = 572 (42), m/z = 592 (44), m/z = 541 (40), m/z = 592/594 (41), m/z = 543 (38), m/z = 563/565 (37), and their main metabolites by Collision Induced Dissociation and in the ion trap using helium as the collision gas with an energy of 35%. MSn experiments were performed in the objective to further characterize the molecular ions corresponding to hydroxylated metabolites. Data acquisition and processing were performed with Xcalibur™ software (version 2.2) with the assistance of Mass Frontier™ software (version 7.0) for proposed fragmentation mechanisms (Thermo Fisher Scientific, France).

## In vivo studies on rats

All animal procedures were in strict accordance with the recommendations of the European Community (council directive 2010/63/EU) and the French National Committee (Decree 2013-118) for the care and use of laboratory animals. The experimental protocols were validated by a local ethics committee for animal use and approved by the French government (n° APAFIS#38612-2022092015556744 v2 and n° APAFIS#28255-2020112314343452 v1). Animals were housed under standard experimental conditions: room temperature (20 ± 2 °C); light/dark cycle (12 h/12 h); water and food *ad libitum* and kept in social groups of at least two rats per cage with enrichment. An acclimation period of 7 days was respected before any experiment.

## AAV-*hP2Y*<sub>12</sub>R animal model

Male Sprague-Dawley rats were stereotactically injected in the right striatum (reference to bregma: AP +0.5mm, R +3mm, V -4.5mm) with 4 µl of AAV-*hP2Y*<sub>12</sub>R (5.0 x 10<sup>9</sup> Vg/µl) under isoflurane anesthesia and buprenorphine treatment (0.05 mg/kg) for pain management. AAV-injected rats were housed for at least one month before in vivo PET imaging or autoradiographic studies.
## Autoradiographic studies

### Heterologous displacement of a specific P2Y₁₂R ligand by compounds 37 and 41.

Autoradiographic studies were performed using 20 µm-thick frozen brain sections from Sprague-Dawley (SD) rats injected stereotactically in the striatum with an adeno-associated viral (AAV) vector expressing the human P2Y₁₂ receptor (*hP2Y₁₂R*). Prior to autoradiography, brain slices were placed at room temperature (RT) during 45 minutes and hydrated in 100 mM Tris-HCl buffer (pH = 7,4) for 15 min. To evaluate the capacity of compounds **37** and **41** to displace specific binding of [³H]PSB-22219 (K_D = 5 nM),²³ a specific tritiated P2Y₁₂ receptor radioligand, adjacent brain slices were incubated for 1 hr at RT with 2 nM [³H]PSB-22219 alone (total binding), or with 1 µM of compounds **37** or **41**. As a reference for non-displaceable binding, brain slices were incubated with 2 nM [³H]PSB-22219 in the presence of 1 µM of AZD1283, a commercially available P2Y₁₂ receptor antagonist (see SI). After incubation, slices were rinsed three times for 5 minutes in ice-cold Tris-HCl buffer, washed in ice-cold distilled water, and dried for 24 h. A high-performance autoradiography imager (β-imager 2000, Biospace Lab, Paris, France) allowing real-time radioactive imaging through direct β-particle counting and absolute radioactivity quantification (detection threshold of 0.001 cpm/mm² for ³H) was used to measure radioactivity in dried tissue sections. A calibration curve was constructed using duplicate standards spotted with known concentrations (from 0.004 to 0.4 Bq in 10 mg) to convert the gray-scale intensity of the autoradiography images into activity per mg. On each individual brain slice of autoradiography images, total binding was segmented based on intensity scaling using PMOD 3.8 software. Next, each region of interest (ROI) was copy-pasted to the adjacent brain slice to evaluate non-specific binding. Activity values were normalized for the surface of the ROI and converted into Bq/mg.

### Homologous displacement: total and non-displaceable binding of compounds 37 and 41.
Frozen brain sections (coronal or axial) from rats injected intrastriatally with AAV-*h*P2Y₁₂R, and from one non-human primate were incubated for 20 minutes with [¹¹C]37 (85 ± 27 MBq, 29 ± 12 GBq/µmol (EOB); 50-389 nM) or [¹⁸F]41 (54 ± 31 MBq, 59 ± 52 GBq/µmol (EOB); 9-16 nM) in Tris Buffer (50mM TRIZMA preset crystals purchased from Sigma-Aldrich) adjusted to pH 7.4 with NaCl. Specific binding was assessed using an excess (10µM) of either unlabeled 37 or 41, respectively. After incubation, slices were rinsed two times for 5 minutes in ice-cold Tris-HCl buffer, washed in ice-cold distilled water. Sections were then dried using an air stream, placed in direct contact with a Phosphor-Imager screen (Molecular Dynamics, Sunnyvale, CA) and exposed for 4 hours. Autoradiograms were analyzed using ImageJ software (https://imagej.nih.gov). Mean gray values for each ROI normalised by area were obtained, and expressed as percentage relative to mean tracer binding on brain sections incubated with the respective radioligand only.

## Immunohistochemistry

Validation of *h*P2Y₁₂ receptor expression in the AAV-*h*P2Y₁₂R injected striatum was performed via immunohistochemistry using antibody against *h*P2Y₁₂R (1:1000; Anaspec; 55043A) diluted in blocking serum solution (3% normal goat serum with 0.2% TritonX-100 in 1X PBS). After three rinses, slides were incubated 1 h with secondary antibody (1:1000; Vector Laboratories; BA1000) diluted in 0.2% TritonX-100 in 1X PBS. To get rid of the excess, slides were rinsed three times and incubated with avidin biotin complex (ABC) (1:250 solution A; 1:250 solution B; Vector Laboratories; PK-6100) in 0.2% TritonX-100 in 1X PBS for 1 h. Slides were washed three times in PBS, before the reveal with tetrachloride diaminobenzidine (DAB) (Vector Laboratories; SK-4100). Before cover slipping, slide mounted tissues were consecutively dehydrated in: 50% ethanol for 1 min, 70% ethanol for 1 min, 95% ethanol for 1 minute two times, 100% ethanol for 1 minute two times, xylene for 3 minutes two times. The images were taken via Epson Scanner,
to have a macroscopic look on the injection site. Immunofluorescent co-labeling of *hP2Y*₁₂*R*, microglia (CD11b) with nuclear counterstain (DAPI) were obtained as follows: brain sections were fixed in 4% PFA for 15 min. PFA blockage was performed with *NH*₄Cl (50 mmol/L) in PBS for 5 minutes and tissue permeabilization was performed with 0.1% Triton in PBS for 5 minutes at RT. Sections were washed with PBS after every step. The sections were saturated with a solution of 5% BSA/0.5% Tween in PBS for 90 minutes at RT to block nonspecific binding. Sections were incubated for 1 hr at RT with primary antibodies in 5% BSA/0.5% Tween in PBS. Sections were stained for *hP2Y*₁₂*R* with rabbit anti-P2Y₁₂R (1:1000; Anaspec, 55043A or Sigma, HPA014518) and for CD11b with rat anti-CD11b (1:500; antibodies-online, #AbIN284101). The sections were then washed (3 × 5 min) in PBS and incubated for 45 minutes at RT with secondary antibodies Alexa Fluor 488 nm goat anti-rabbit IgG and Alexa Fluor 546 nm goat anti-rat IgG (1:1000; Invitrogen, #A32731 and #A-11081, respectively) in 5%BSA/0.5% Tween 0.5% in PBS, and then washed again (3 × 5 min) in PBS. Specificity of the antibodies was tested using controls omitting the first antibodies. Sections were mounted with a 4,6-diamidino-2-phenylindole (DAPI) Prolong Antifade Kit (Molecular Probes, Life Technologies) for nuclear counterstain. Fluorescence image acquisition was performed with an AxiObserver Z1 microscope (Zeiss, Germany).

## Biodistribution of [¹⁸F]41 in healthy rats

12 healthy Sprague Dawley male rats (282 ± 17 g) were injected via the tail vein with [¹⁸F]41 (26 ± 8 MBq). The animals, placed under deep gaseous isoflurane/oxygen anesthesia (4%), were sacrificed by decapitation at 5, 15, 30 and 60 minutes after injection (n = 2 per time point). Blood, plasma and organs (brain, cerebellum, heart, liver, spleen, kidney, pancreas, stomach, intestine, lung, bone marrow, bone (femur) and muscle) were collected, weighed and counted for radioactivity using a cross-calibrated gamma counter (Cobra Quantum D5003; Perkin-Elmer,
France). All values were corrected for fluorine-18 decay (109.8 minutes) till the injection time.
The concentration of radioactivity per gram of tissue in whole blood, plasma and tissues were
normalized by the injected dose and expressed as a percentage of injected dose per gram of tissue
± standard deviation (%ID/g ± SD). The tissue concentrations were corrected by plasma
concentration at the same time (%ID/g tissue/%ID/g plasma).

**In vivo metabolism of [¹⁸F]41**

Blood samples were collected with 5 mL hemolysis tubes using a heparinized syringe (at 5, 15, 30 and 60 minutes after injection), and they were immediately centrifuged at 4 °C for 5 minutes at 3000 g to obtain cell-free plasma. Plasma samples (500 µL) were deproteinated with 700 µL of acetonitrile, and centrifuged at 4 °C for 2 minutes (3000 g). The whole supernatants were analyzed by radio-HPLC following the procedure described below. Simultaneously, the brain was excised, the cerebellum was dissected. Both cerebral hemispheres and the cerebellum were weighted and assessed for radioactivity. One hemisphere was homogenized by sonication in 1 mL of acetonitrile on ice and was centrifuged at 3500 rpm for 5 min. The pellet was re-suspended and further sonicated in 1 mL of acetonitrile. After centrifugation of the second homogenate, both extracts were pooled before analysis by radio-HPLC. The radioactivity in the resulting pellets (plasma and brain) was measured to calculate the recovery of radioactivity in acetonitrile. The whole supernatants (plasma and brain extracts) were diluted in an equal volume of water and the whole samples were injected onto the radio-HPLC system consisted of a quaternary gradient pump, an ASI100T autosampler and a UVD170U UV-Vis detector (Dionex S.A., France) online with a LB-509 radioisotope detector (MX Z500 cell; Berthold, France). [¹⁸F]41 and its radiometabolites were separated using a 5µm, 10x250 mm, C18 Atlantis T3 preparative column (Waters, France). The mobile phases consisted of 0.1% v/v trifluoroacetic acid in water (A) and 0.1% v/v trifluoroacetic
acid in acetonitrile (B). A linear gradient from 10% to 100% of B in 12 minutes was applied to the column, at a flow rate of 5 mL/min. The thermostatic column oven was set at 25°C. Data acquisition and processing were performed with Chromeleon software (version 1.0; Dionex). The radioactivity due to non-metabolized [¹⁸F]41 in plasma or in brain was expressed as a fraction of the total radiolabeled peak areas. The percentages of non-metabolized [¹⁸F]41 in plasma or in the brain as a function of time were fitted by a nonlinear regression analysis (OriginPro software version 8.5; OriginLab, Northampton, MA) using a standard following biexponential decay equation.

## In vivo PET imaging

In vivo PET imaging was performed under isoflurane anesthesia (2-2.5% in O₂) in healthy (n = 8) or AAV-hP2Y₁₂R injected rats (n = 7). A catheter was inserted into the caudal tail vein and anesthetized animals were rapidly transferred to the microPET scanner. PET images were acquired on a Siemens Inveon® small animal PET-CT or PET scanner. Both scanners were used in parallel in order to assure a high specific radioactivity at the time of injection. [¹¹C]37 or [¹⁸F]41 were injected intravenously (19.0 ± 5.9 MBq and 31.4 ± 14.3 MBq, respectively) and dynamic PET acquisition (60 min) started with radioligand injection. The efflux substrate behavior of radiolabeled 37 or 41 was investigated in the presence or absence of P-glycoprotein and breast cancer resistance protein inhibitor tariquidar (8 mg/kg) administered intravenously 15 minutes before PET as previously described.³¹

## PET data analysis.

PET images were reconstructed with the Fourier rebinning algorithm and the three-dimensional ordered-subset expectation-maximization algorithm, including normalization, attenuation, scatter, and random corrections. Image analysis was performed using PMOD software (version 3.8,
PMOD Technologies Ltd., Zurich, Switzerland). Brain PET images were corrected for scatter, radioactive decay, injected dose and body weight. Brain distribution of [¹¹C]37 and [¹⁸F]41 was expressed as standardized uptake values (SUV), with SUV = tissue activity (kBq/cc)/[injected dose (kBq)/body weight (g)]. The PET images were then spatially co-registered to the Schiffer rat brain PET template for delineation of brain regions. Using the predefined VOIs of the template, SUV time activity curves (TACs) of different regions (whole brain, striatum, and cerebellum) were extracted from the images. TACs for all animals injected were verified and n = 2 animals excluded from the study due to non-intravenous injection.

*In vivo experiments in non-human primate.*

**NHP.** one male cynomolgus monkey (*Macaca fascicularis*, 10.4 kg, 7 years). The animal use procedure was in accordance with the recommendations of European regulations (EU Directive 2010/63) and was approved by the local ethical committee (CETEA n°44) and the French Ministry of Education and Research (APAFIS #40586-2023013115557146 v1). The experimental data reported in this study comply with the ARRIVE (Animal Research: Reporting In Vivo Experiments) guidelines.

**PET experiment.** [¹¹C]37 was formulated in 0.9% aqueous saline with 3% ethanol (v/v). The radiochemical purity of [¹¹C]37 was greater than 98%, and the molar activity at the time of injection was 61.5 GBq/µmol. PET imaging was performed on a Biograph (Siemens) under standard anesthesia and monitoring procedures.³² Briefly, the macaque received a mixture of ketamine (10 mg/kg, i.m.) and xylazine (0.5 mg/kg) to induce anesthesia. After intubation in the supine position, venous catheters were inserted for radiotracer injections (saphenous vein) and propofol infusion (saphenous vein). Another catheter was inserted into the femoral artery for arterial blood sampling. The head of the macaque was positioned under the camera before
administration of a 2 mL IV bolus of propofol, followed by a 1 mL/kg/h IV infusion under oxygen ventilation. Dynamic PET data acquisition (90 min) started with the IV bolus injection of [¹¹C]37 (223.6 MBq/kg, 1.12 µg/kg). During PET acquisition, a total of 25 blood samples of 0.3 mL were collected: 15 samples during the first 20 minutes, followed by 7 samples every 10 minutes. Larger samples (1–1.5 mL) were collected at 5, 15, 30, 60, and 90 minutes for metabolite analysis.

**Blood measurement and analysis.** Plasma was separated from whole blood by centrifugation (5 minutes, 2054xg, 4 °C), and 50 µL of plasma and whole blood were counted using a PET cross-calibrated gamma well counter (WIZARD2, PerkinElmer, France) to obtain the whole-blood and plasma activity curves. All data were corrected for radioactive decay from the time of injection. For larger blood samples, 500 µL of plasma was deproteinized with acetonitrile. The supernatant was injected into a high-performance liquid chromatography system equipped with an Atlantis® T3 5 µm 10 × 250 mm column (Waters) and an Atlantis® T3 5 µm 3.9 × 10 mm pre-column (Waters), with an LB-514 radioactivity flow detector (Berthold, La Garenne Colombes, France, MX Z100 cell). The parent fraction of [¹¹C]37 was calculated as a percentage of the total radioactivity (metabolites and parent). A 1-exponential decay function was fitted to the parent fraction of [¹¹C]37, which was time-multiplied with the plasma activity curve to obtain the metabolite-corrected arterial plasma input function.

**PET data analysis.** PET images were reconstructed using a standard PSF-OSEM-3D algorithm, with corrections for radioactive decay, scatter, attenuation, and detector inhomogeneity. PET image analysis was performed using PMOD software version 4.3 (PMOD Technologies Ltd., Zurich, Switzerland). After co-registration of PET-MR images, a cynomolgus atlas published by Ballanger and coworkers³³ was normalized to PET images to extract time activity curves in different brain regions and expressed as SUV ([radioactivity per mL of plasma/injected
radioactivity] × body weight). Volumes of interest (VOIs) included the whole brain, frontal cortex, cingulate cortex, temporal cortex, orbitofrontal cortex, parietal cortex and cerebellum. Kinetic modeling was conducted using Logan graphical analysis with the arterial plasma input function corrected for radiometabolites to derive the volume of distribution (VT; mL/cm³) in each region under all tested conditions.

## ASSOCIATED CONTENT

### Supporting Information.

The data associated to this research work (experimental procedures for intermediate compounds preparation, radiochemistry experiments, details for in vitro (affinity, metabolism) and in vivo experiments, 1H and 13C NMR spectra, HPLC chromatograms of tested ligands) are available in the online supplementary file (PDF).

## AUTHOR INFORMATION

### Corresponding Author

*Mylène Richard: mylene.richard@cea.fr

### ORCID Numbers

Marie-Anne Peyronneau: 0000-0003-1520-4666

Margot Corbel: 0000-0001-7297-2448

Sébastien Goutal: 0000-0001-7797-0740

Maud Goislard: 0000-0002-8705-345X

Haneen Al Hroub: 0000-0002-2033-0344

Marie Théry: 0009-0003-4494-4564

Sébastien Beuché: 0009-0006-2203-8060
Hashem Ali M Al Musawi: 0000-0003-0664-4942

Fabien Chauveau: 0000-0002-4177-741X

Nadja Van Camp: 0000-0002-2261-7314

Alexis Bemelmans: 0000-0001-7605-5225

Christa E. Müller: 0000-0002-0013-6624

Bertrand Kuhnast: 0000-0002-5035-4072

Alexandra Winkeler: 0000-0003-3397-2570

Mylène Richard: 0000-0003-0305-578X

**Present Address**

† CisBio international, 91400 Saclay, France

**Author Contributions**

AW, NVC, BK, MAP, CEM and MR contributed to the design of the study and oversaw the research project. EP, MT, HAMAM and SB carried out the chemistry and characterization of the compounds. MAP carried out the metabolic experiments on cold compounds. EP and HAH performed the biological evaluation of the unlabeled ligands. EP optimized the radiosyntheses.

NVC and AB established the animal model of P2Y₁₂R overexpression. MAP, CD, AW & EP performed the biodistribution and metabolic experiments with radioligands. AW, NVC, HB, CD, SG & MG carried out the autoradiography and TEP imaging experiments and images reconstruction. MR, MAP, NVC and AW wrote the paper with contributions from all coauthors.

All authors have read and commented on the manuscript and given approval to the final version of the manuscript.

**Acknowledgments**
This work was funded by Agence Nationale de la Recherche (under grants ANR-21-CE18-0067-01, project "PURImaging" and ANR-11-INBS-0006, project "France Life imaging"), the German Research Foundation (DFG, MU 1665/12-1, project number 471773752) and CEA "Bottom up" grant VISIOGLIE. This work was partly carried out on a platform of the France Life Imaging network (grant ANR-11-INBS-0006). This project has received financial support from the CNRS through the MITI interdisciplinary programs. The authors thank Julien Varin and Maxime Le Failler for cyclotron operation and carbon-11 and fluorine-18 production and Cyril Colas from the "Fédération de Recherche" ICOA/CBM (FR2708) for HRMS analyses. The authors gratefully acknowledge the help and input of Françoise Hinnen for automation of carbon-11 labeling. HAMAM is grateful for a PhD scholarship from the government of Saudi-Arabia.

## Notes

The authors declare no competing interests.

*During the reviewing process, a paper describing a novel P2Y<sub>12</sub>R radioligand was published.*<sup>34</sup>

## ABBREVIATIONS

AAV: adeno-associated viruses; BBB: brain blood barrier; CDI: carbonyldiimidazole; CNS: central nervous system; DMF: *N,N*-dimethylformamide; EOB: end of bombardment; ESI: electrospray ionisation; HLM: human liver microsomes; HPLC: high pressure liquid chromatography; HRMS: high resolution mass spectrometry; IC<sub>50</sub>: half-maximal inhibitory concentration; ID: injected dose; LC/MS: liquid chromatography / mass spectrometry; MS: multiple sclerosis; NADPH: nicotamide adenine dinucleotide phosphate; NHP: non-human primate; NMR: nuclear magnetic resonance; PET: positron emission tomography; RLM: rat liver microsomes; ROI: region of interest; SD: standard deviation; SD: sprague dawley; SEM:
standard error of the mean; SUV: standardized uptake value; TAC: time-activity curve; TQD:

tariquidar; TSPO: translocator protein 18 kDa; UV: ultraviolet; VOI: volume of interest

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Graphical Abstract

<figure><img src="image_22.png" /><figcaption>PET imaging of [<sup>18</sup>F]41 in healthy rats</figcaption></figure>

<figure><img src="image_23.png" /></figure>

* Homogeneous binding in AAV-hP2Y<sub>12</sub>R rat models

* Displacement in blocking conditions

* Low crossing of the BBB

* Efflux substrate behaviour of [<sup>18</sup>F]41