Synthesis and evaluation of highly selective quinazoline-2,4-dione ligands for sphingosine-1-phosphate receptor 2

Abstract

A series of twenty-nine new quinazoline-2,4-dione compounds were synthesized and their IC₅₀ values for binding toward sphingosine-1-phosphate receptor 2 (S1PR2) were determined using a [³²P]S1P binding assay. Seven compounds 2a, 2g, 2h, 2i, 2j, 2k, and 5h exhibit high S1PR2 binding potencies (IC₅₀ values < 50 nM) and four of these new compounds 2g, 2i, 2j, and 2k have IC₅₀ values (<10 nM) of 6.3, 5.7, 4.8, and 2.6 nM, and are highly selective for S1PR2 over other S1PR subtypes, S1PR1, 3, 4, and 5. Compounds 2a and 2i were chosen for C-11 radiosynthesis through O-[¹¹C]methylation of precursors 13 and 2k with good radiochemical yields (35–40%), high chemical and radiochemical purity (>98%), and high molar activity (153–222 GBq μmol⁻¹, at the end of bombardment). [¹¹C]2a and [¹¹C]2i were further evaluated by the ex vivo biodistribution study. The results showed that both tracers have low brain uptake, preventing their potential for neuroimaging application. Further explorations of this class of S1PR2 PET tracers in peripheral tissue diseases are underway.

New quinazoline-2,4-dione analogues were developed as sphingosine 1-phosphate receptor 2 ligands. [¹¹C]2i has great potential to serve as a positron emission tomography probe in peripheral tissue diseases.

Introduction

Sphingosine-1-phosphate (S1P) is a lipid signaling molecule that is generated from sphingosine by activation of sphingosine kinase 1 (SphK1) or sphingosine kinase 2 (SphK2).¹ S1P plays an important role as an intracellular second messenger and acts extracellularly through five specific cell surface G protein-coupled receptors, named S1PR1–5.^2–4 Among these receptors, sphingosine-1-phosphate receptor 2 (S1PR2), originally named as endothelial differentiation G-protein coupled receptor 5 (EDG-5), is extensively expressed in various tissues, and it can activate a range of intracellular signaling cascades.^5,6 Studies find that S1PR2 couples with Gi, Gq, and G_12/13 family proteins and activation of S1PR2, in particular, is associated with an array of cellular functions and pathologies, including modulating neuronal excitability,⁷ hepatocyte regeneration,⁸ vascular permeability functions,^9,10 and the survival of pancreatic β-cells.^11,12 To investigate the function of S1PR2, investigators have explored the effects of its inactivation in the female SJL experimental autoimmune encephalomyelitis (EAE) murine model of multiple sclerosis (MS),^13,14 streptozotocin (STZ)-induced diabetic rats,^15,16 and carbon tetrachloride (CCl₄) or cholestasis-induced liver fibrosis.^17,18

The modern positron emission tomography (PET) imaging technique has allowed researchers to in vivo and in situ noninvasively visualize, characterize, and quantify biological processes at the cellular or molecular level.^19,20 Particularly, PET with a suitable radiotracer, enables the functional imaging analysis of biological processes with high sensitivity, a well-recognized complementary modality to anatomic imaging modalities such as magnetic resonance imaging (MRI) and computerized tomography (CT), widely used for preclinical and clinical applications. Today, PET is widely used for early diagnosis, prediction, monitoring of diseases, guiding the development of therapeutics, and assessing target protein engagement.^21,22 Therefore, PET with a specific S1PR2 radiotracer may advance our understanding of S1PR2 pathophysiologic functions in diseases. However, this is hampered by the lack of novel S1PR2 PET radiotracers. The natural ligand, S1P, which has a high binding affinity with S1PR1–5 at the nanomolar level, was first radiolabeled with isotope P-32 or P-33. [³²P]S1P and [³³P]S1P (Fig. 1) are mainly used for screening the potencies of compounds binding toward S1PR1–5,^23,24 but their applications are limited due to their low commercial availability and high cost. We had reported [¹²⁵I]TZ6544 (Fig. 1), which is easily made and used in the laboratory for determining the IC₅₀ values of new compounds binding toward S1PR2 using a radioactive competitive binding assay. We also reported that [¹²⁵I]TZ6544 is able to detect an increase of S1PR2 expression in the kidney of diabetic rats induced by streptozotocin (STZ). To date, [¹¹C]TZ34125 (Fig. 1) was the only reported S1PR2 PET tracer and a PET study with [¹¹C]TZ34125 indicated the sexual dimorphism of S1PR2 expression in the cerebellum of cyclosporin A treated SJL mice.¹⁴ However, its inability to penetrate the blood–brain-barrier (BBB) prevented its implementation in central nervous system (CNS) diseases. Therefore, identification of a novel S1PR2 PET tracer is imperative for PET quantitative measurement of S1PR2 in CNS disorders and other diseases. We had reported several S1PR2 ligands (Fig. 1) with high binding affinity and selectivity for S1PR2.^25,26 For example, TZ5732 is an aryloxybenzene analogue with a high binding affinity to S1PR2 (IC₅₀ = 14.6 nM).²⁵ Although it is possible that radiosynthesis of [¹⁸F]TZ5732 may be achieved by starting with its corresponding borate precursor, we had not successfully obtained the desired radiochemical yield and purity. Herein, we reported our efforts on the design and synthesis of a new series of S1PR2 ligands and in vitro determination of their potency for S1PR2. We also performed the C-11 radiosynthesis of two potent tracers, [¹¹C]2a and [¹¹C]2i, and ex vivo evaluation of [¹¹C]2a and [¹¹C]2i to test the feasibility of quantifying S1PR2 expression in tissues of rodents.

Fig. 1. The structures of known S1PR2 radiotracers and potent S1PR2 compounds.

Results and discussion

Chemistry

Compound 1 was reported as a potent S1PR2 ligand for treating inflammatory and allergic conditions in animals through targeting S1PR2.²⁷ Therefore, our efforts had focused on structural optimization of compound 1 to develop new S1PR2 compounds. We expect that our structural modification will lead to the discovery of new S1PR2 ligands with high potency and selectivity, which can be radiosynthesized using conventional C-11 or F-18 radiochemistry methods for further in vivo evaluation in animals. The syntheses of new target compounds were carried out by following Schemes 1–3. Firstly, we modified the carboxylic acid group in the structure of compound 1 using carboxylic acid amides to prepare new compounds 2a–k as shown in Scheme 1. Compound 1 was made according to the literature²⁷ and served as a key intermediate. Using 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5b]pyridinium 3-oxid hexafluorophosphate (HATU) and N,N-diisopropylethylamine (DIPEA) as coupling agents at room temperature (RT), compound 1 reacted with different substituted amines to generate compounds 2a–k.

Scheme 1. Syntheses of 2a–k. Reagents and conditions: (a) amines, HATU, DIPEA, dimethylformamide, RT.

Scheme 3. Syntheses of 8a–e and 9a–e. Reagents and conditions: (a) amines, triethylamine, ethyl acetate, reflux; (b) triphosgene, DIPEA, dichloromethane, 0 °C–RT; (c) 2-bromo-1-(3-chloro-4-ethoxyphenyl)ethan-1-one, K₂CO₃, dimethylformamide, RT; (d) 2-bromo-N-(5-chloro-2,4-dimethoxyphenyl)acetamide, K₂CO₃, dimethylformamide, RT.

Secondly, we focused on the modification of the 5-chloro-2,4-dimethoxyphenyl motif of compound 2g, in which the carboxylic acid in compound 1 was replaced with carboxylic acid N-(2-methoxyphenyl) amide. Eight new compounds 5a–h were synthesized as shown in Scheme 2. Compound 3 was made according to a reported method.²⁸ The subsequent condensation with o-anisidine afforded the key intermediate 4, followed by coupling with different substituted bromides to yield compounds 5a–h.

Scheme 2. Syntheses of 5a–h. Reagents and conditions: (a) o-anisidine, HATU, DIPEA, dichloromethane, RT; (b) bromides, K₂CO₃, dimethylformamide, RT.

Thirdly, the carboxylic acid group in compound 1 was replaced using different substituted alkyl chains. Ten new compounds 8a–e and 9a–e were synthesized as shown in Scheme 3. Generally, commercially available isatoic anhydride (6) was reacted with different substituted amines and further cyclized using triphosgene to afford compounds 7a–e. The following nucleophilic substitution reaction of 7a–e with 2-bromo-1-(3-chloro-4-ethoxyphenyl)ethan-1-one or 2-bromo-N-(5-chloro-2,4-dimethoxyphenyl)acet-amide yielded the target compounds 8a–e or 9a–e, respectively.

In vitro binding assay

After all the new compounds were purified and characterized, their S1PR2 binding potencies were determined. The IC₅₀ values for JTE-013, literature reported compound 1, and the newly synthesized ligands were determined through the radioactive competitive binding assay with [³²P]S1P that was freshly made in our lab following a reported procedure.²⁴ It was observed that the IC₅₀ values of JTE-013 and compound 1 were 66.8 nM and 21.3 nM, respectively, indicating that compound 1 is over 3-fold more potent than JTE-013 for S1PR2. The new analogues generated from structural modification of compound 1 were expected to be potent toward S1PR2. Our in vitro binding data of these new compounds 2a–k, 5a–h, 8a–e, and 9a–e toward S1PR2 are presented in Table 1.

The binding potencies (IC₅₀ ± SD) of 2a–k, 5a–h, 8a–e, and 9a–e toward S1PR2^a.

Compound	IC50a (nM)	Compound	IC50a (nM)
2a	40.1 ± 7.0	2b	91.4 ± 17.6
2c	204 ± 96	2d	376 ± 91
2e	175 ± 13	2f	58.7 ± 9.0
2g	6.3 ± 0.9	2h	39.1 ± 7.0
2i	5.7 ± 0.5	2j	4.8 ± 0.5
2k	2.6 ± 0.3	5a	54.9 ± 6.0
5b	>1000	5c	>1000
5d	>1000	5e	>1000
5f	>1000	5g	287 ± 69
5h	49.8 ± 4.9	8a	>1000
8b	>1000	8c	>1000
8d	>1000	8e	>1000
9a	>1000	9b	>1000
9c	>1000	9d	>1000
9e	>1000

^aIC₅₀ values were determined from at least two independent experiments, each run was performed in duplicate; for compounds with IC₅₀ < 100 nM, at least three independent experiments were performed, each run was performed in duplicate.

As described above, for compounds 2a–k, the carboxyl acid group in the structure of compound 1 was replaced using different substituted amides. Compounds 2a–f were synthesized to investigate the impact of different alkyl amide side chains on the S1PR2 binding affinity. As shown in Table 1, compound 2a, possessing a terminal hydroxyethyl group, showed a good binding affinity with an IC₅₀ value of 40.1 nM for S1PR2; when using two and three polyethylene glycol (PEG) units to extend the ethoxyl side chain to generate compounds 2b and 2c, the IC₅₀ values for S1PR2 were further increased to 91.4 and 204 nM, respectively. Compound 2d or 2e has an N-methyl carboxyl amide or N,N-dimethyl carboxyl amide group, and they did not show favorable S1PR2 binding with IC₅₀ values of 376 and 175 nM, respectively. Compound 2f, having a 3-ethylpyridine side chain, showed moderate binding activity with an IC₅₀ value of 58.7 nM. To investigate the impact of the substituted aryl amide functional group on the S1PR2 binding affinity, compounds 2g–k were studied. Compared to the alkyl amides 2a–f, all the substituted aryl amides 2g–k are more potent than 2a–f. Particularly, compounds 2g, 2h, and 2i showed high binding potencies with IC₅₀ values of 6.3, 39.1, and 5.7 nM, respectively, indicating that the methoxy group at both ortho (2g) and para positions (2i) is more favorable to bind toward S1PR2 than the methoxy group at the meta position (2h). After demethylation, the generated phenols 2j and 2k, with the hydroxyl group at ortho- and para- positions, improved the S1PR2 binding affinity to 4.8 and 2.6 nM, respectively.

Since the aryl amide group showed favorable binding affinity toward S1PR2, further modifications were focused on replacing the N-(5-chloro-2,4-dimethoxyphenyl)acetamide motif with different aryl groups, and compounds 5a–h were synthesized and their IC₅₀ values for binding toward S1PR2 were determined. As shown in Table 1, compounds 5b–g were inactive for S1PR2, but compounds 5a and 5h, having N-(5-chloro-2-methoxyphenyl)acetamide and 1-(3-chloro-4-ethoxyphenyl)ethan-1-one motifs, showed moderate potencies with IC₅₀ values of 54.9 and 49.8 nM, respectively, suggesting that the chloride at the 5-position of the aromatic ring was critical for S1PR2 binding.

Based on the structure–activity relationship analysis of compounds 2a–k and 5a–h, we found that the N-(5-chloro-2,4-dimethoxyphenyl)acetamide motif in 2a–k and 1-(3-chloro-4-ethoxyphenyl)ethan-1-one motif in 5h have a chlorine atom at the aromatic ring which might be favorable for S1PR2 binding. Therefore, our further design and synthesis of new compounds retained at least one of these two motifs. We replaced the N-2-acetic acid group with N-alkyl groups to generate compounds 8a–e and 9a–e. Our in vitro data showed that all compounds 8a–e and 9a–e lost the binding affinity for S1PR2 with IC₅₀ > 1000 nM, suggesting that the N-alkyl chains replacing the N-2-acetic acid group would prevent the compounds' binding toward S1PR2.

As is known, S1P receptors have five subtypes (S1PR1–5), and selective binding toward S1PR2, but not the other S1P receptors, is an important character for a promising S1PR2 compound. Therefore, the highly potent compounds 2a, 2g–k, and 5h were further screened for their binding potencies toward S1PR1, 3, 4, and 5. As shown in Table 2, all these S1PR2 potent compounds exhibited low binding activity toward S1PR1, 3, 4, and 5 (IC₅₀ values > 1000 nM), indicating that compounds 2a, 2g–k, and 5h were highly selective for S1PR2 versus other S1PR subtypes.

The binding affinity of potent compounds toward S1PR1, 3, 4, and 5^a.

Compound	IC50 (nM)
	S1PR2	S1PR1	S1PR3	S1PR4	S1PR5
S1Pb	3.6 ± 0.5	1.4 ± 0.3	0.4 ± 0.2	151 ± 82	3.1 ± 1.1
2a	40.1 ± 7.0	>1000	>1000	>1000	>1000
2g	6.3 ± 0.9	>1000	>1000	>1000	>1000
2h	39.1 ± 7.0	>1000	>1000	>1000	>1000
2i	5.7 ± 0.5	>1000	>1000	>1000	>1000
2j	4.8 ± 0.5	>1000	>1000	>1000	>1000
2k	2.6 ± 0.3	>1000	>1000	>1000	>1000
5h	49.8 ± 4.9	>1000	>1000	>1000	>1000

^aIC₅₀ values were determined from at least two independent experiments, each run was performed in duplicate.

^bRef. 11.

Based on the encouraging in vitro results, some of these highly potent and selective S1PR2 compounds have high potential to be therapeutic drug candidates, and some of them are worthy of being selected for further in vivo evaluation of their suitability to be PET tracers for investigating S1PR2 expression in animals.

Radiosynthesis of [¹¹C]2a and [¹¹C]2i

Because compound 2a was the first potent S1PR2 ligand we found in this series of derivatives, the carbon-11 labeled [¹¹C]2a was radiosynthesized and pilot evaluation was performed. Later, our in vitro binding results showed that compounds 2g and 2i–k have high binding affinity with IC₅₀ values less than 10 nM, indicating that they are more potent candidates than 2a. Considering that the structures of compounds 2g and 2i–k are very similar, [¹¹C]2i was selected as a representative radiotracer for radiosynthesis and further evaluation in animals.

The synthesis of precursor 13 and [¹¹C]2a is shown in Scheme 4. The condensation of compound 3 with ethanolamine yielded intermediate 10, followed by coupling with tert-butyl bromoacetate to afford compound 11. After deprotection of the tert-butyl group of compound 11, the resulting acid 12 was coupled with 4-amino-2-chloro-5-methoxyphenol to generate the precursor 13. The radiosynthesis of [¹¹C]2a was accomplished by O-[¹¹C]methylation of the phenol group in compound 13 using [¹¹C]CH₃I at 90 °C under basic conditions in dimethyl sulfoxide (DMSO). The radioactive product was purified using a reverse-phase HPLC column combined with solid-phase extraction (SPE). The final dose was formulated using a 10% ethanol saline solution. An aliquot dose sample was taken for quality control using an analytical HPLC system. The radiotracer [¹¹C]2a was authenticated by co-injection with cold reference standard 2a, and the chemical and radiochemical purity were also determined. The radiochemical yield of [¹¹C]2a was ∼35%, the molar activity was ∼153 GBq μmol⁻¹ (at the end of the bombardment, EOB, n = 3), and the chemical and radiochemical purity of [¹¹C]2a was >99%. The total synthetic time including isolation and formulation was ∼45 min.

Scheme 4. Syntheses of [¹¹C]2a and [¹¹C]2i. Reagents and conditions: (a) ethanolamine, HATU, DIPEA, dichloromethane, RT; (b) tert-butyl bromoacetate, K₂CO₃, dimethylformamide, RT; (c) trifluoroacetic acid, dichloromethane, RT; (d) 4-amino-2-chloro-5-methoxyphenol, HATU, DIPEA, dimethyl sulfoxide, RT; (e) [¹¹C]CH₃I, 5 M NaOH, DMSO, 90 °C.

The radiosynthesis of [¹¹C]2i was performed using a similar radiolabeling method of making [¹¹C]2a, except that the final dose of [¹¹C]2i was formulated using 10% of ethanol in 5% Kolliphor® Macrogol 15-hydroxystearate (HS-15) solution because of the low solubility of [¹¹C]2i in 10% ethanol saline solvent. HS-15 is used as a non-ionic solubilizing agent to increase the solubility of [¹¹C]2i. The radiosynthesis of [¹¹C]2i was achieved with a good radiochemical yield of ∼40%, high molar activity of ∼222 GBq μmol⁻¹ (at EOB, n = 10), and high chemical and radiochemical purity of >98% determined by an analytic HPLC system. The total synthetic time including isolation and formulation was ∼40 min.

Evaluation of [¹¹C]2a and [¹¹C]2i in rodents

All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Washington University and approved by the Institutional Animal Care and Use Committee (IACUC) of Washington University in St. Louis, Missouri, USA.

Ex vivo biodistribution of [¹¹C]2a in SD rats

To quickly assess the tissue distribution of [¹¹C]2a, particularly to check if it can penetrate the BBB, [¹¹C]2a was administrated into adult male Sprague Dawley (SD) rats by tail intravenous (IV) injection. The rats were euthanized at 5, 30, and 60 min post-injection of [¹¹C]2a, and the tissues were collected, weighed, and counted to calculate the radioactivity uptake (% ID per gram). The tissue distribution of [¹¹C]2a in adult male SD rats is presented in Table 3. At 5 min post-injection, the uptake of the blood, heart, lung, pancreas, spleen, kidney, liver, thymus, and brain was 0.27, 0.38, 0.31, 0.66, 0.29, 1.83, 1.08, 0.21, and 0.02, respectively. [¹¹C]2a had a rapid clearance from the blood, heart, lung, pancreas, spleen, kidney, liver, and thymus, and the uptake in these tissues was only 0.05, 0.08, 0.06, 0.09, 0.05, 0.27, 0.16, and 0.08, respectively at 30 min post-injection. The high initial uptake in the pancreas, kidney, and liver may result from the high expression of S1PR2 protein in these tissues,^11,18,29 suggesting that [¹¹C]2a has a high possibility to be used for investigating S1PR2 expression in these peripheral tissues. Nevertheless, the lower initial brain uptake (ID per gram) of [¹¹C]2a of 0.02 prevents its use for investigating S1PR2 expression in the brain for CNS disorders with intact BBB function. In addition, the moderate IC₅₀ value (40.1 nM) for S1PR2 suggested that further structural optimization of [¹¹C]2a is necessary to identify a suitable S1PR2 radiotracer for the neuroimaging study. Therefore, we focused on the in vivo evaluation of [¹¹C]2i with an IC₅₀ value of 5.7 nM, a more potent S1PR2 radiotracer than [¹¹C]2a.

Tissue distribution of [¹¹C]2a in adult male SD rats (n = 4 for each time point).

Tissues	Tissue uptake (% ID per gram)
	5 min	30 min	60 min
Blood	0.27 ± 0.05	0.05 ± 0.02	0.03 ± 0.01
Heart	0.38 ± 0.06	0.08 ± 0.02	0.03 ± 0.01
Lung	0.31 ± 0.05	0.06 ± 0.02	0.03 ± 0.01
Pancreas	0.66 ± 0.12	0.09 ± 0.03	0.05 ± 0.01
Spleen	0.29 ± 0.05	0.05 ± 0.02	0.02 ± 0.01
Kidney	1.83 ± 0.38	0.27 ± 0.10	0.16 ± 0.06
Liver	1.08 ± 0.28	0.16 ± 0.05	0.10 ± 0.04
Thymus	0.21 ± 0.08	0.08 ± 0.06	0.04 ± 0.01
Brain	0.02 ± 0.01	0.01 ± 0.00	0.01 ± 0.00

Ex vivo biodistribution of [¹¹C]2i in SJL mice

Increased expression of S1PR2 in disease-susceptible CNS regions of female SJL mice and female MS patients was reported,¹³ and our PET study of [¹¹C]TZ34125 also suggested the sexual dimorphism of S1PR2 expression in the cerebellum of the SJL mice.¹⁴ However, [¹¹C]TZ34125 had challenges in crossing the blood-brain-barrier to measure the S1PR2 expression directly in the CNS. Considering that 2i is a very potent and selective S1PR2 ligand, we expected that the radiotracer [¹¹C]2i could penetrate the BBB. We decided to use SJL mice for the tissue distribution study of [¹¹C]2i because the SJL strain mouse is susceptible to experimental autoimmune encephalomyelitis (EAE) for multiple sclerosis research. Meanwhile, we want to make a parallel comparison of [¹¹C]2i with [¹¹C]TZ34125 using the same strain of mice. We carried out the tissue distribution of [¹¹C]2i using normal female SJL mice (8 weeks). Animals (n = 4 per group) were euthanized at 5, 30, and 60 min post-injection. The biodistribution data is presented in Table 4. At 5 min post-injection, the uptake of the blood, heart, lung, pancreas, spleen, kidney, liver, thymus, and brain was 5.10, 4.47, 154.5, 30.02, 8.24, 58.72, 1.48, and 0.59, respectively. The female SJL mice brain uptake of [¹¹C]2i reached ∼0.6 at 5, 30, and 60 min post-injection, which is still relatively lower than [¹¹C]TZ34125 (0.78 ID% g⁻¹ at 30 min post-injection on SJL female mice¹⁴), suggesting that [¹¹C]2i has limited penetration of the BBB in SJL mice. Therefore, [¹¹C]2i may have limited utility for investigating CNS diseases without BBB disruption. Potential applications of [¹¹C]2i will thus be focused on the disease of peripheral tissues.

The tissue distribution of [¹¹C]2i in SJL female mice (8 weeks old, n = 4 per group).

Tissues	Tissue uptake (% ID per gram)
	5 min	30 min	60 min
Blood	5.10 ± 0.67	1.23 ± 0.13	1.00 ± 0.25
Heart	4.47 ± 1.10	1.13 ± 0.13	0.89 ± 0.10
Lung	154.5 ± 37.8	22.87 ± 7.29	14.46 ± 2.16
Pancreas	2.77 ± 0.27	1.72 ± 0.48	2.03 ± 0.56
Spleen	30.02 ± 5.80	20.75 ± 1.66	14.21 ± 3.03
Kidney	8.24 ± 1.37	3.10 ± 0.41	2.23 ± 0.18
Liver	58.72 ± 10.04	57.89 ± 7.92	45.43 ± 6.30
Thymus	1.48 ± 0.21	0.97 ± 0.16	0.70 ± 0.13
Brain	0.59 ± 0.07	0.57 ± 0.05	0.62 ± 0.15

Conclusions

In summary, we successfully explored twenty-nine new S1PR2 ligands and discovered seven compounds 2a, 2g–k, and 5h with high S1PR2 binding affinity (IC₅₀ < 50 nM) and high selectivity for S1PR2 (IC₅₀ > 1000 nM for S1PR1, 3, 4, and 5). Compounds 2a and 2i were selected for C-11 radiosynthesis, and both [¹¹C]2a and [¹¹C]2i were made with good radiochemical yields, high chemical and radiochemical purity, and high molar activity. The preliminary tissue distribution studies indicated that [¹¹C]2a and [¹¹C]2i had initial high uptake in the lung, pancreas, kidney, and liver, consistent with the S1PR2 expression in tissues, but low uptake in the brain of rodents. Further studies of [¹¹C]2i will focus on the evaluation of its potential in the disease of peripheral tissues other than the CNS.

Conflicts of interest

There are no conflicts to declare.