Design and Synthesis of Novel PET Radiotracers for Imaging Sphingosine-1-Phosphate Receptor 5 (S1PR5) in the Brain

Jiawen Lang; Wenjuan Zhou; Tianyu Huang; Hao Jiang; Hong Chen; Shuxian Xu; Lin Qiu; Madison Heady; Yanbo Yu; Matthew R Brier; Ying-Hwey Nai; Caroline Guglielmetti; Joel S Perlmutter; Zhude Tu

doi:10.1021/acs.jmedchem.5c00881

. Author manuscript; available in PMC: 2026 Jun 3.

Published in final edited form as: J Med Chem. 2025 Jun 6;68(12):12991–13018. doi: 10.1021/acs.jmedchem.5c00881

Design and Synthesis of Novel PET Radiotracers for Imaging Sphingosine-1-Phosphate Receptor 5 (S1PR5) in the Brain

Jiawen Lang ^a, Wenjuan Zhou ^a, Tianyu Huang ^a, Hao Jiang ^a, Hong Chen ^a, Shuxian Xu ^a, Lin Qiu ^a, Madison Heady ^a, Yanbo Yu ^a, Matthew R Brier ^b, Ying-Hwey Nai ^a, Caroline Guglielmetti ^a, Joel S Perlmutter ^a,^b,^c, Zhude Tu ^a

PMCID: PMC13225913 NIHMSID: NIHMS2179144 PMID: 40476381

Abstract

Sphingosine-1-phosphate receptor-5 (S1PR5) is highly expressed in oligodendrocytes, plays an important role in neurodegenerative disorders like multiple sclerosis. We designed, synthesized, and determined binding potencies of 27 novel S1PR5 ligands. Four radiotracers [¹¹C]7, [¹⁸F]7a, [¹¹C]12a, and [¹⁸F]12b were synthesized for characterization of their in-vitro and in-vivo binding properties. [¹⁸F]7a had good rat brain uptake with 0.62 %ID/g at 5 min, while other three tracers had lower brain uptake. [¹⁸F]7a had K_d values of 2.2, 4.6, 27.6 nM for recombinant human S1PR5 cell membrane, the C57BL/6 mouse brain, and the human cerebral cortex respectively. Pretreatment with S1PR5 potent modulators effectively impacted the rodent brain uptake of [¹⁸F]7a. Cuprizone-fed mice had reduced [¹⁸F]7a brain uptake, from loss of oligodendrocytes and decreased S1PR5 expression. [¹⁸F]7a showed good brain uptake and retention in macaque, no radiometabolites entered the rat brain, further supporting [¹⁸F]7a as a promising radiotracer for imaging S1PR5 in the brain.

Keywords: Multiple sclerosis, Sphingosine-1-phosphate receptor 5, Carbon-11 radioligand, Flourine-18 radioligand, PET imaging, Cuprizone mouse model, Oligodendrocytes

INTRODUCTION

Sphingosine-1-phosphate receptors (S1PRs) are a five-family member G-protein-coupled receptors, activated by their natural bioactive lysophospholipid ligand, sphingosine-1-phosphate (S1P).^1–3 S1P signaling plays a pivotal role in regulating cellular processes, such as cell survival, trafficking, growth, and differentiation in various cell types within different systems including the immune, cardiovascular, central nervous system (CNS), and others.^4–7 Each S1P receptor has its unique expression pattern in different tissues and responds to diseases through its specific pathway.^{4, 8–10} Among the five S1PRs family members, S1PR5 is predominantly expressed in CNS and immune systems and it plays an important role in autoimmune diseases, neurodegenerative disorders, and carcinogenesis.^11–13 In the CNS, S1PR5 promotes the survival and differentiation of mature oligodendrocytes, which are essential for myelin maintenance and nerve function.^14–16 Notably, S1PR5 has been implicated in multiple sclerosis (MS), a chronic autoimmune disease characterized by demyelination and neuroinflammation. In the cuprizone model of MS, which mimics the pathogenesis of demyelination and remyelination in MS, S1PR5 is believed to contribute to oligodendrocyte survival and myelin repair, making it a promising therapeutic target for MS treatment.^{17, 18} Furthermore, S1PR5 activation in brain endothelial cells enhances barrier integrity and reduces the transendothelial migration of monocytes in vitro, suggesting that S1PR5 could help mitigate the neuroinflammatory processes associated with MS.^19–22 Additionally, S1PR5 has been implicated in other disorders associated with blood-brain barrier disruption, such as Alzheimer’s, Huntington’s, and Crohn’s disease.^{12, 23, 24} In the immune system, S1PR5 regulates natural killer (NK) cell migration under both homeostatic and inflammatory conditions, which may be implemented in cancer therapy.²⁵ The non-selective drug FTY720 (Fingolimod) is potent to all S1P receptor subtypes except S1PR2 and can induce internalization of S1PR, causing a downregulation of lymphocytes and makes targeting S1P signaling as an attractive therapeutic strategy.^{26, 27} To date, the precise role and contribution of S1PR5 to pharmacological effects from FTY720 or the second generation of S1P signaling modulators such as Ozanimod remains to be illustrated.^28–30

Several compounds, including FTY720, Siponimod, Ozanimod, and A-971432 have been used to investigate the modulatory effect of S1PR5 although these ligands have their limitations including weak selectivity for S1PR5 over S1PR1 and other subtypes, unoptimized pharmacokinetic properties, challenges in synthesis of their C-11 or F-18 labeled counterparts.^31–34 The lack of suitable S1PR5-specific ligands and radiotracers hampers systemic understanding of the mechanism of S1PR5 modulation in human and animal models of diseases. In 2025, Morgan et al. reported [¹⁸F]TEFM78 as an S1PR5 PET tracer with an EC₅₀ of 22.3 nM (Figure 1). This tracer exhibited good brain uptake in rodents but showed rapid washout within 20 minutes indicating inadequate brain retention.³⁵ This highlights the need for improved S1PR5 PET tracers with better pharmacokinetics. Despite these challenges, PET imaging with promising S1PR5 radiotracers will offer a unique noninvasive methodology to visualize and quantify S1PR5 expression changes in vivo for precise assessment of S1PR5-related diseases in the early stage and monitoring the efficacy of treatment targeting S1PR5.

Figure 1. — The structures of 1 ([¹⁸F]TEFM78), four radiotracers [¹¹C]7, [¹⁸F]**7a,** [¹¹C]**12a,** and [¹⁸F]**12b.**

Recently, a new S1PR5 ligand, compound 7, was reported as a highly potent and selective S1PR5 antagonist with EC₅₀ value of 0.1 nM, determined using a calcium mobilization assay.^{36, 37} This assay measures changes in intracellular calcium levels upon receptor activation, indirectly reflecting the functional activity of the S1P receptor. Compound 7 has good brain penetration and oral bioavailability that facilitate its potent inhibitory effect on NK cell migration toward S1PR5.^{37, 38} To develop novel S1PR5 ligands and PET radiotracers, herein, we designed, synthesized, and evaluated 27 new derivatives sharing the naphthalene pharmacophore with compound 7. Based on our radioactive competitive binding affinity assay using [³²P]S1P radioligand, nine compounds demonstrated both good S1PR5 potency (IC₅₀ < 125 nM) and selectivity (IC₅₀ > 1000 nM for S1PR1–4), as shown in Table 2. To test the feasibility of PET imaging S1PR5 in vivo in animals, we selected four compounds for C-11 or F-18 radiolabeling to make four radiotracers [¹¹C]7, [¹⁸F]7a, [¹¹C]12a, and [¹⁸F]12b (Figure 1). We performed rodent biodistribution studies, PET imaging in cuprizone fed mouse model of MS, PET brain imaging in nonhuman primates, and HPLC radiometabolite analysis in rodents and nonhuman primates. Our data show [¹⁸F]7a is the most promising S1PR5 radiotracer based on the following considerations: a) good blood brain barrier (BBB) permeability and high brain uptake; b) no defluorination happened in vivo; c) good in vivo metabolic stability with no brain-penetrant radiometabolites to confound PET measurements; d) our direct saturation binding study showed that [¹⁸F]7a had K_d value of 2.173 ± 0.795 nM for recombinant human S1PR5 cell membrane, K_d and the B_max value of 4.6 ± 1.1 nM, and 427.2 ± 38.5 fmol/mg for C57BL/6 mouse brain tissue, 27.6 ± 3.6 nM and 381.6 ± 10.7 fmol/mg for human cerebral cortex respectively, further confirming the high binding potency of [¹⁸F]7a toward the S1PR5; e) Our PET study showed that cuprizone fed mice (after 4 weeks on diet) had decreased brain uptake of [¹⁸F]7a compared to normal control mice. Immunohistology studies confirmed that lower brain S1PR5 expression in the cuprizone fed mouse compared to normal diet mouse led to decreased brain uptake of [¹⁸F]7a; f) the lower brain S1PR5 expression correlated with lower number of oligodendrocyte cells, and the reduced expression of myelin basic protein in cuprizone-fed mice, a widely used model for studying the demyelination pathogenesis of MS. These findings suggest that PET with [¹⁸F]7a can effectively detect S1PR5 expression changes in the brain of the cuprizone mice compared to the normal control mice. Together, our data suggest that [¹⁸F]7a is a promising radiotracer for quantifying S1PR5 expression in CNS for neurological disorders, particularly in MS.

Table 2.

Binding potency and selectivity of newly synthesized compounds for S1PR5^a


Compound	R¹, R², R³, or R⁴	IC₅₀ (nM)
Compound	R¹, R², R³, or R⁴	S1PR5	S1PR1	S1PR2	S1PR3	S1PR4
7	R¹ = Me	9.40±2.20	>1000	>1000	>1000	>1000
7a	R¹ = F	85.4±6.55	>1000	>1000	>1000	>1000
12a	R² = OMe	28.2±3.11	>1000	>1000	>1000	>1000
12b	R² = OCH₂CH₂F	61.5±5.1	>1000	>1000	>1000	>1000
16a		3.28±1.34	>1000	>1000	>1000	>1000
16b		124.0±11.5	>1000	>1000	>1000	>1000
16g		2.07±0.19	>1000	>1000	>1000	>1000
16j		0.38±0.02	>1000	>1000	>1000	>1000
19f		88.0±5.90	>1000	>1000	>1000	>1000

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IC₅₀ values were determined in at least 3 independent experiments, each run was performed in duplicate; assays for compounds that showed weak binding potency (IC₅₀ > 1000 nM) were only repeated twice.

RESULTS AND DISCUSSION

Chemistry:

Based on our structural analysis of the reported potent S1PR5 compound 7,³⁷ we divided the molecule into four sections: A, B, C, and D as in Figure 2. We systematically modified each section to design and synthesize new S1PR5 compounds.

Following Scheme 1, we first replaced the methyl group at the 4-position of the naphthalene ring (Fragment A) of lead compound 7 with either fluorine (F-) or methoxy (-OCH₃) groups. Commercially available starting material 2 was reacted with cis-4-(tert-butyl)cyclohexyl methanesulfonate in the presence of Cs₂CO₃, affording intermediate 3 in 43% yield. This compound was subsequently converted to methyl-substituted 4 in 89% yield using ZnMe₂ under Pd(dppf)Cl₂-catalyzed reflux conditions. The fluorine derivative 4a was synthesized using NFSI under −78 °C condition in 83% yield, and the methoxy-substituted compound 4b was obtained through several reactions involving B₂Pin₂, NaBO₃·4H₂O, and CH₃I, with 33% yield. After introducing an aldehyde group at the 1-position of the naphthalene ring provided compounds 5, 5a-b, their tertiary amines 6, 6a-b were made through reductive amination reaction with moderate yields (50−53%). Hydrolysis of 6, 6a-b afforded the targeted S1PR5 ligands 7, 7a-b respectively.

As shown in Scheme 2, we introduced different substituents at position B (position 7 of the naphthalene ring) of lead compound 7 to explore if new substituted groups at position B can retain or improve the S1PR5 binding potency. Starting with compound 8 (see the supporting information), an aldehyde group was introduced at the para position of the 4-methyl group, and the bromine substituent was converted to a boron ester using palladium catalysis to afford intermediate 9 with a yield of 46%. Oxidation of the boron ester 9 with sodium perborate tetrahydrate produced the hydroxyl-substituted compound 10 with 42% yield. This intermediate was then transformed into methoxy-substituted compound 11a (78% yield) or fluorine ethoxy-substituted compound 11b (84% yield) through substitution reaction under basic conditions. Compounds 11a and 11b underwent reductive amination and hydrolysis to obtain ligands 12a and 12b with 12% and 24% yields, respectively, as shown in Scheme 2A. Additionally, the fluorine-substituted product 13 was obtained from compound 8 using NFSI, with an 81% yield, followed by the introduction of an aldehyde group to form product 14 with 58% yield, which was converted into fluorine-substituted S1PR5 ligand 12c by a two-step procedure with a yield of 31% as shown in Scheme 2B.

Scheme 2 A&B. — Synthesis of target compounds **12a-c**. Reagents and reaction conditions: (a) SnCl₄, CHCl₂OCH₃, CH₂Cl₂, 0 °C−rt, 4 h; (b) B₂Pin₂, Pd(dppf)Cl₂, KOAc, 1,4-dioxane, 95 °C, 9 h; (c) NaBO₃·4H₂O, THF/H₂O, rt, 2 h; (d) CH₃I, K₂CO₃, DMF, rt, overnight; or 1-bromo-2-fluoroethane, K₂CO₃, CH₃CN, 80 °C, 8 h; (e) ethyl piperidine-4-carboxylate, ethyl acetate, NaBH(OAc)₃, 60 °C, overnight; (f) LiOH·H₂O, MeOH/THF/H₂O, rt, 3 h; (g) ethyl piperidine-4-carboxylate, methanol, 70 °C, NaBH₃CN, overnight.

Our next strategy was to focus on introducing different types of substituents (R³) at fragment C (the position 1 of naphthalene ring) of compound 7, as shown in Scheme 3. Starting from compound 5, we synthesized a series of acid-containing compounds 16a-f with moderate to good yields (25−60%). Compound 16a was prepared by reacting aliphatic amine salts with compound 5 via reductive amination reaction at room temperature with DIPEA added. Using the same method, we obtained compound 16b with a methyl substituent at the alpha position of the propionic acid. Compound 16c with a fluorine substituent at the alpha position of the propionic acid, was obtained under reflux condition. Additionally, a methyl group was introduced into the N-H substituent, yielding ligands 16d-f. We also synthesized the target compounds 16g-p with different alcohols, sulfonic acid, sulfonamide, fluorine-substituted amines, and cyclic amines as shown in Scheme 3A to investigate the impact of different substituents on Fragment C. Furthermore, we modified fragment D (position 2 of naphthalene ring) of compound 7 to develop new S1PR5 analogues as shown in Scheme 3B by replacing the tert-butyl-cyclohexyl motif with methyl- or fluorine-substituted aliphatic chains, obtaining compounds 19a-f. Key intermediates 17a-f were reacted through reductive amination with ethyl 3-aminopropanoate salts to produce secondary amine esters 18a-f with 48–60% yields, and subsequently hydrolyzed to obtain S1PR5 ligands 19a-f with 52–81% yields.

Scheme 3 A-B. — Synthesis of target compounds **16a-p** & **19a-f**. Reagents and reaction conditions: **16a-b**: (a) i: amine regents, 1,2-DCE, DIPEA, NaBH(OAc)₃, rt, overnight; ii: LiOH·H₂O, MeOH/THF/H₂O, rt, 3 h; **16c**: amine regent, DIPEA, CH₃COOH, NaBH₃CN, EtOH, 80 °C, overnight; **16d-f**: (i) amine regents, 1,2-DCE, DIPEA, NaBH(OAc)₃, rt, overnight; ii: Formaldehyde (36.5–38% in H₂O), CH₃COOH, MeOH, NaBH₃CN, rt, overnight; iii: LiOH·H₂O, MeOH/THF/H₂O, rt, 3 h; **16g-i** and **16m**: amine regents, 1,2-DCE, NaBH(OAc)₃, rt, overnight; **16j**: amine regent, CH₃COOH, NaBH₃CN, EtOH, 80 °C, overnight; **16k, l, n,** and **18a-f**: amine regents, 1,2-DCE, DIPEA, NaBH(OAc)₃, rt, overnight; **16o-p**: amine regents, 1,2-DCE, NaBH(OAc)₃, 45 °C-rt, overnight; TFA, DCM, 0 °C-rt, 2 h; Formaldehyde (36.5–38% in H₂O), CH₃COOH, MeOH, NaBH₃CN, rt, overnight (**16o**); 1-bromo-2-fluoroethane, K₂CO₃, CH₃CN, 80 °C, 8 h (**16p**); **19a-f**: (c) LiOH·H₂O, MeOH/THF/H₂O, rt, 3 h.

In vitro determination of binding activity.

The binding potencies of our newly synthesized S1PR5 ligands and reference compound 7 toward S1PR5 were determined using our well-established [³²P]S1P competitive binding assay³⁹. The binding affinity (IC₅₀ values) of all tested compounds towards recombinant human S1PR5 cell membrane preparation were shown in Table 1. Our data showed that the binding potency toward S1PR5 was decreased for ligands when the 4-methyl group in the structure of compound 7 was replaced with fluorine or methoxy group. The fluorine-containing compound 7a had a moderate IC₅₀ of 85.4 ± 6.55 nM in our radioligand competitive binding assay, whereas the IC₅₀ value of compound 7 was determined as 9.40 ± 2.20 nM. Notably, our IC₅₀ value is approximately ~94-fold higher than the reported EC₅₀ value of 0.1 nM; the differential potency values are possibly due to the difference between two types of assays used. Our assay uses radiolabeled S1P to directly measure the binding potency of S1P receptor in IC₅₀ value, whereas the reported EC₅₀ value uses a calcium mobilization assay to indirectly measure the S1P receptor activity, which can be affected by other factors rather than directly measuring the interaction between S1P and S1PR5.³⁶ We expect the potency of 7a would be higher using the reported calcium mobilization screening assay to determine its EC₅₀ value for S1PR5. Similarly, we expect that our other newly synthesized compounds would have higher potency using the reported calcium mobilization screening assay. Directly comparing two screening assays would help further develop and evaluate new S1PR5-potent radiotracers. Based on our current radioligand competitive binding assays to determine the IC₅₀ values of S1P ligands, the methoxy-containing compound 7b had an IC₅₀ value of >1000 nM, indicating a significant loss of potency toward S1PR5. The significant differences in IC₅₀ values for compounds 7, 7a, and 7b, highlight the critical role of the 4-methyl group in retaining the high S1PR5 binding potency, and guided us to retain the methyl group for further structural optimizations. Consequently, we kept the 4-methyl group in the naphthalene moiety, and modified position B by replacing the H atom with methoxy or fluoroethoxy group to generate 12a and 12b with IC₅₀ values of 28.2 ± 3.11 nM and 61.5 ± 5.1 nM respectively. These results provide encouraging prospects for developing them as potential S1PR5 tracers. However, the fluorine-substituted compound 12c showed reduced potency (IC₅₀ = 199.9 ± 36.6 nM). Our next exploration focused on replacing the N-containing cyclic acid in Fragment C using different open alkyl chain acid, compound 16a showed excellent binding potency with IC₅₀ = 3.28 ± 1.34 nM. Introducing a methyl or fluorine group in the alpha-position of propanoic acid decreased potency with IC₅₀ values of 124.0 ± 11.5 nM and 252.3 ± 34.3 nM for 16b and 16c. We next used a methyl group to replace the proton (H-) at linkage N-atom to make corresponding ligands 16d, 16e, and 16f. However, these three compounds had significantly reduced binding potency towards S1PR5 with IC₅₀ values of 348.4 ± 28.8, 474.6 ± 27.4 and 503.9 ± 28.7 nM for 16d, 16e and 16f, respectively, suggesting the importance of the proton (H-) at the N-atom for maintaining high S1PR5 potency. Therefore, we decided to retain the second amino group (NH) and replace the carboxylic acid group in Fragment C using different substituted groups; the sulfonic acid, alcohol, thiomethyl, and sulfonamide-substituted amine moiety were introduced to generate compounds 16g-n. Our data showed that 16g and 16j were the two most potent S1PR5 ligands with IC₅₀ values of 2.07 ± 0.19 nM and 0.38 ± 0.02 nM, respectively. Whereas compounds 16h, 16i, 16l, and 16n had moderate binding potency with IC₅₀ values of 300.0 ± 66.0, 323.8 ± 61.5, 515.9 ± 3.0, and 312.4 ± 43.5 nM, respectively. In contrast, compounds 16k & 16m completely lost binding potency with IC₅₀ values > 1000 nM. Additionally, compounds 16o and 16p were made by introducing a cyclized 1,4-piperazinyl moiety to replace the second amine with an alkyl chain, but both 16o & 16p lost binding potency toward S1PR5 with IC₅₀ values > 1000 nM. Our last optimization strategy focuses on Fragment D by replacing the 4-tert-butyl-cyclohexyl group in the structure of compound 7 with methyl or fluorinated alkanes having a 2–6 carbon chain (-CH₂-) and a fluorine atom at the terminal of the carbon chain, resulting in compounds 19a, 19b, 19c, 19d, 19e, and 19f. Surprisingly, only compound 19f with six carbon chain (-CH₂-) and fluorine at the chain terminal showed moderate binding potency with an IC₅₀ value of 88.8 ± 5.90 nM, all other five compounds 19a-e lost binding potency for S1PR5 with IC₅₀ value > 900 nM. Together, we successfully synthesized twenty-seven new compounds along with the reference compound 7. Our in vitro binding affinity data revealed that nine of these compounds have good binding potency to human S1PR5 protein with IC₅₀ value < 125 nM. These findings provide critical insights into the structural features necessary for high S1PR5 binding affinity and provide information for future design and synthesis of potent and selective S1PR5 PET radiotracers for both preclinical and clinical applications.

Table 1.

Binding potencies of newly synthesized compounds for S1PR5^a


Compound	R¹	IC₅₀ (nM)	Compound	R²	IC₅₀ (nM)
7	Me	9.40±2.20	12a	OMe	28.2±3.11
7a	F	85.4±6.55	12b	OCH₂CH₂F	61.5±5.1
7b	OMe	>1000	12c	F	199.9±36.6

Compound	R³	IC₅₀ (nM)	Compound	R³	IC₅₀ (nM)
16a		3.28±1.34	16i		323.8±61.5
16b		124.0±11.5	16j		0.38±0.02
16c		252.3±34.3	16k		>1000
16d		348.4±28.8	16l		515.9±3.0
16e		474.6±27.4	16m		>1000
16f		503.9±28.7	16n		312.4±43.5
16g		2.07±0.19	16o		>1000
16h		300.0±66.0	16p		>1000

Compound	R⁴	IC₅₀ (nM)	Compound	R⁴	IC₅₀ (nM)
19a	Me	>1000	19d		>900
19b		>1000	19e		>1000
19c		>1000	19f		88.8±5.90

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IC₅₀ values were determined in at least 3 independent experiments; each run was performed in duplicate. Assays for compounds that showed no activity (IC₅₀ > 400 nM) were only repeated twice.

To evaluate the selectivity of the nine potent compounds for S1PR5 versus other S1P receptor subtypes 1, 2, 3, and 4, we next tested their potency against cell membranes for those S1P receptor subtypes (Table 2). Our in vitro binding assay showed that ligands 7, 7a, 12a-b, 16a-b, 16g, 16j, and 19f had IC₅₀ values > 1000 nM for recombinant human S1PR1, 2, 3, and 4 cell membranes, suggesting that these nine ligands, and potentially all their analogues, are selective for S1PR5. In order to evaluate the possibility of developing a novel S1PR5 radiotracer from the potent and selective S1PR5 ligands, compounds 7, 7a, 12a, and 12b were selected for F-18 or C-11 radiolabeling to make [¹¹C]7, [¹⁸F]7a, [¹¹C]12a, and [¹⁸F]12b.

Molecular Docking Studies.

To investigate the structure and bioactivity relationship, molecular docking analyses were conducted for the interaction of compound 7a with S1PR5 protein (PDB code: 7YXA³¹). As shown in Figure 3, compound 7a significantly overlapped with the native co-crystallized ligand ONO-5430608³¹ (a known inverse selective S1PR5 agonist) and exhibited similar binding interactions with the protein (Figures 3B and 3C). Further analysis revealed that the bulky naphthalene ring of 7a occupied an allosteric subpocket formed by the non-conserved hydrophobic residues Cys43, Leu292, Gly293, and Met296. In addition, 7a orchestrated a notable hydrogen bond interaction between the NH₂ group of Lys24 and both oxygen atoms of the carboxylic acid. The molecular simulation studies in conjunction with cell assays provide evidence of the high binding potency of 7a against S1PR5.

Figure 3. — The protein-ligand docking study and visualization. A) The overall structure of human S1PR5 (PDB: 7YXA) and docking box. B) Pose of co-crystallized ONO-5430608 (purple). **C&D**) 3D and 2D schematic diagrams of intermolecular interactions of the predicted binding modes of 7a with S1P5 (PDB code: 7YXA). Hydrogen bonds are highlighted in yellow and green dotted lines separately for 3D and 2D structures, the receptor is shown in gray or cyan. The molecular visualization was generated using PyMOL and Discovery Studio Visualizer.

Radiochemistry.

To synthesize radiotracers [¹¹C]7, [¹⁸F]7a, [¹¹C]12a, and [¹⁸F]12b, the corresponding precursors were designed and synthesized by following Scheme 4. A reductive amination reaction of compound 20 afforded the tertiary amine compound 21. Then, a cross-coupling reaction between the compound 21 and B₂pin₂, catalyzed by palladium (II) metal afforded the boron eater 22, the precursor for making [¹¹C]7 and [¹⁸F]7a. Additionally, through a reductive amination reaction of compound 10 afforded 23, the precursor for both [¹¹C]12a and [¹⁸F]12b.

The radiosyntheses of the four radiotracers [¹¹C]7, [¹⁸F]7a, [¹¹C]12a, and [¹⁸F]12b were accomplished by following Scheme 5. Both [¹¹C]7 and [¹⁸F]7a were made by starting with its boronic ester precursor 22. For the C11-labeling of [¹¹C]7, we explored various conditions to achieve optimal results, finding that both the base and temperature were crucial for a good radiochemical yield (showed in Table S1 of the supplementary information). Among the different base agents, sodium hydroxide solution (5N) proved to be the most effective. Additionally, high temperatures led to the decomposition of the precursor, and we found that 60 °C was the optimal temperature for the C11-labeling procedure. [¹¹C]7 synthesis was achieved by trapping [¹¹C]CH₃I in a reaction vessel containing Pd₂dba₃ and P(o-toyl)₃ dissolved in DMF. After trapping, oxidative addition intermediates reacted with precursor 22 under NaOH (5N, 3 μL) at 60 °C, followed by hydrolysis using NaOH (5N, 55 μL) to remove the ethyl group, yielding [¹¹C]7 with moderate radiochemical yield (11–14%, n > 4), high chemical purity (>95%), and radiochemical purity (>99%), and high molar activity (>40.7 GBq/μmol, decay corrected to end of bombardment, EOB). For [¹⁸F]7a, a copper-mediated nucleophilic ¹⁸F-fluorination of aryl boronic esters 22 with [¹⁸F]KF under heating condition was employed, followed by hydrolysis using sodium hydroxide solution (5N) to afford the F-18 labelled [¹⁸F]7a with a good radiochemical yield (22~29%, n > 4), high chemical and radiochemical purity (>99%), and high molar activity (>55.6 GBq/μmol) at the end of synthesis (decay corrected to EOS). The radiochemistry yield was improved using a solvent mixture of 1-butanol and N,N-dimethylacetamide. Using the same base and reaction temperature of making [¹¹C]7, [¹¹C]12a was accomplished by O-[¹¹C]methylation of its naphthol precursor 23 with [¹¹C]CH₃I under basic condition, followed by hydrolysis using NaOH (5N) to remove the ethyl group afforded [¹¹C]12a with moderate radiochemical yield (11–14%, n > 6), high chemical purity (>90%), and radiochemical purity (>99%), and high molar activity (>54 GBq/μmol, decay corrected to the end of bombardment, EOB).

The radiosynthesis of [¹⁸F]12b was accomplished by a two-pot procedure. First, 1,2-bis(tosyloxy)ethane 24 reacted with [¹⁸F]KF to produce 2-[¹⁸F]fluoroethyl tosylate [¹⁸F]25 with a reliable yield (55 ± 3%, n > 10, decay corrected to EOS). Second, the naphthol precursor 23 was reacted with [¹⁸F]25 in DMSO in the presence of 5N NaOH solution to provide a radiochemical yield of ~20% for [¹⁸F]12b, >90% chemical purity and >99% radiochemical purity, and molar activity of ~50 GBq/μmol (n = 3, decay corrected to EOS). The final injection dose for in vitro and in vivo biological studies was formulated using 10% ethanol in saline. An aliquot sample of injection dose was authenticated using an analytical HPLC system for quality control.

Ex vivo biodistribution study of [¹¹C]7, [¹⁸F]7a, [¹¹C]12a and [¹⁸F]12b in Sprague-Dawley rats.

To check the brain uptake and peripheral tissue biodistribution of these four radiotracers, studies were performed in adult male Sprague-Dawley (SD) rats (Table 3). At 5 min post-injection, these four radiotracers showed relatively high uptake in organs including lung, liver, spleen, kidney, heart, and pancreas as shown in Table 3: [¹¹C]7 showed uptake values of 2.15, 3.80, 1.50, 2.74, 2.79, and 1.47 (%ID/g) in these organs, respectively; [¹⁸F]7a displayed values of 3.22, 7.50, 2.61, 4.81, 4.90, and 2.77 (%ID/g), [¹¹C]12a had values of 5.26, 4.02, 1.84, 2.60, 2.83, and 1.16 (%ID/g), and [¹⁸F]12b exhibited values of 3.33, 5.54, 1.86, 3.27, 3.46, and 1.47 (%ID/g), respectively. These results indicate a great potential for imaging S1PR5 expression in peripheral tissues with our novel S1PR5 radioligands. The radioactivity quickly washed out from lung, liver, spleen, kidney, and heart with much lower uptake in these organs 30 minutes post-injection. Notably, its F-18 labeled counterpart [¹⁸F]7a showed a high brain uptake (%ID/g) of 0.62, 0.52, 0.55, and 0.54 at 5, 30, 60, and 120 minutes, respectively (Table 3). For [¹¹C]7, [¹¹C]12a, and [¹⁸F]12b, the brain uptakes (%ID/g) were relatively low at 5 minutes with 0.15, 0.12 and 0.04, respectively. Among these four radiotracers, [¹⁸F]7a had good brain penetration, uptake, and brain retention from 5 min to 120 min, making it a promising radiotracer for imaging S1PR5 in the CNS. In addition, no significant amount and trend of bone uptake were observed for [¹⁸F]7a from 5 min to 120 min, and [¹⁸F]12b from 5 min to 60 min, indicating that both F-18 radiotracers [¹⁸F]7a and [¹⁸F]12b have no in vivo defluorination. Together, our biodistribution study in SD rats suggests that [¹⁸F]7a is the most promising S1PR5 radiotracer for imaging S1PR5 in the CNS and worth pursuing PET imaging study in vivo in animals.

Table 3.

Biodistribution of [¹¹C]7, [¹⁸F]7a, [¹¹C]12a, and [¹⁸F]12b in Male Sprague Dawley Rats

[¹¹C]7^a				[¹⁸F]7a^b
Organs	5 min	30 min	60 min	5 min	30 min	60 min	120 min
blood	0.16±0.03	0.13±0.01	0.10±0.01	0.27±0.03	0.15±0.02	0.14±0.01	0.12±0.02
lung	2.15±0.44	0.98±0.10	0.83±0.01	3.22±0.31	1.31±0.22	1.29±0.08	1.13±0.26
liver	3.80±0.75	2.20±0.12	1.96±0.06	7.50±1.82	2.75±0.47	2.68±0.25	2.29±0.46
spleen	1.50±0.34	0.71±0.03	0.56±0.05	2.61±0.67	0.96±0.15	0.90±0.03	0.81±0.18
kidney	2.74±0.37	1.43±0.24	1.17±0.12	4.81±0.93	1.59±0.29	1.55±0.05	1.30±0.37
muscle	0.24±0.05	0.36±0.04	0.42±0.02	0.20±0.04	0.49±0.05	0.55±0.03	0.50±0.12
fat	0.11±0.02	0.16±0.03	0.18±0.02	0.09±0.02	0.28±0.05	0.30±0.02	0.28±0.05
heart	2.79±0.29	1.17±0.16	0.95±0.04	4.90±0.63	1.60±0.22	1.55±0.13	1.35±0.25
pancreas	1.47±0.21	1.14±0.21	0.84±0.10	2.77±0.66	1.31±0.31	1.21±0.08	0.94±0.35
brain	0.15±0.02	0.18±0.02	0.19±0.02	0.62±0.13	0.52±0.09	0.55±0.03	0.54±0.14
brain stem	0.20±0.03	0.25±0.07	0.23±0.01	0.66±0.14	0.60±0.10	0.70±0.07	0.73±0.15
bone	na^d	na^d	na^d	0.69±0.26	0.32±0.04	0.35±0.03	0.28±0.10
[¹¹C]12a^c				[¹⁸F]12b^e
blood	0.13±0.08	0.08±0.08	0.08±0.01	0.21±0.01	0.20±0.04	0.16±0.01
lung	5.26±1.10	2.75±1.31	1.52±0.60	3.33±0.34	2.40±0.26	2.32±0.91
liver	4.02±0.72	2.20±0.35	1.89±0.23	5.54±0.92	3.75±0.13	3.20±0.31
spleen	1.84±0.35	0.72±0.13	0.65±0.09	1.86±0.18	1.18±0.04	0.99±0.10
kidney	2.60±0.31	1.21±0.10	1.15±0.22	3.27±0.33	2.16±0.25	1.83±0.13
muscle	0.12±0.02	0.22±0.04	0.24±0.05	0.24±0.04	0.48±0.05	0.58±0.05
fat	0.05±0.02	0.10±0.01	0.09±0.01	0.15±0.05	0.23±0.03	0.25±0.03
heart	2.83±0.30	1.01±0.04	1.00±0.13	3.46±0.31	2.19±0.39	1.68±0.20
pancreas	1.16±0.23	0.83±0.02	0.87±0.11	1.47±0.21	1.64±0.27	1.63±0.15
brain	0.12±0.02	0.12±0.02	0.12±0.02	0.04±0.00	0.05±0.00	0.05±0.00
brain stem	0.19±0.04	0.16±0.06	0.14±0.03	0.06±0.01	0.06±0.01	0.06±0.01
bone	na^d	na^d	na^d	0.37±0.03	0.43±0.08	0.38±0.05

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%ID g⁻¹ values (mean ± SD) with n = 4 rats per group (~260 ± 20 gram).

%ID g⁻¹ values (mean ± SD) with n = 4 rats per group (~210 ± 10 gram).

%ID g⁻¹ values (mean ± SD) with n = 4 rats per group, rat weight: ~280 ± 20 gram.

not applicable.

%ID g⁻¹ values (mean ± SD) with n = 4 rats per group, rat weight: ~210 ± 10 gram.

In vitro direct binding determination of K_d and B_max values of [¹⁸F]7a.

To determine the binding equilibrium dissociation constant (K_d) value of [¹⁸F]7a toward S1PR5, we performed a saturation binding assay directly with [¹⁸F]7a on recombinant human S1PR5 cell membrane protein. The non-specific binding was determined by adding 10 μM of the cold reference compound 7 (Figure 1). [¹⁸F]7a exhibited a K_d value of 2.173 ± 0.795 nM (R² = 0.987) toward recombinant human S1PR5 cell membrane as shown in Figure 4A. In addition, we performed saturation autoradiography analysis of [¹⁸F]7a on brain tissue sections from 12-week-old male C57BL/6 mice, and postmortem normal human cerebral brain tissues. The radioactivity uptake in the brain was fitted to a non-linear regression one-site saturation binding model. Our data revealed that [¹⁸F]7a had a K_d value of 4.6 ± 1.1 nM and a Bmax value of 427.2 ± 38.5 fmol/mg for C57BL/6 mouse brain tissues, and a K_d value of 27.6 ± 3.6 nM and a Bmax value of 381.6 ± 10.7 fmol/mg for human cerebral cortex tissues, as shown in Figure 4B–E. Our in vitro binding affinity characterization of [¹⁸F]7a with recombinant human S1PR5 membrane and brain tissues of C57BL/6 mouse and postmortem human brain further confirmed the potency of [¹⁸F]7a toward S1PR5, particularly for CNS S1PR5. Additionally, saturation autoradiography studies on rodent and human brain tissues also showed high Bmax values of [¹⁸F]7a, indicating great potential for imaging CNS S1PR5 with our novel radioligand.

Figure 4. — *In vitro* evaluation of binding affinity of [¹⁸F]7a. (A) Saturation binding assay of [¹⁸F]7a toward recombinant human S1PR5 cell membrane, [¹⁸F]7a has a K_d of 2.173 ± 0.795 nM with a R² of 0.987. (B&C) Saturation autoradiograph assay of [¹⁸F]7a in C57BL/6 mouse brain tissue, the K_d and Bmax values were 4.6 ± 1.1 nM, and 427.2 ± 38.5 fmol/mg. (D&E) Saturation autoradiograph assay of [¹⁸F]7a in human cerebral cortex tissue, the K_d and the Bmax value of 27.6 ± 3.6 nM and 381.6 ± 10.7 fmol/mg respectively.

PET/CT imaging studies in the C57BL/6 mice at baseline and pretreatment with S1PR5 modulators.

To check the tracer brain uptake and in vivo binding specificity for S1PR5, we performed PET/CT studies of [¹⁸F]7a under different conditions using a Mediso nanoScan PET/CT scanner. Male C57BL/6 mice (12 weeks old, ~30 g) were divided into four groups: 1) baseline condition (n =13); 2) pretreatment with a potent S1PR5 ligand 16g, synthesized by our group (Table 1) (4 mg/kg, n = 2); 3) pretreatment with A-971432, a specific S1PR5 agonist (4 mg/kg, n = 3); and 4) pretreatment with ozanimod, a clinical use S1PR1 and S1PR5 dual modulator (4 mg/kg, n = 3). For each group, S1PR5 potent compound was administrated intravenously (IV) 5 min prior to the injection of [¹⁸F]7a. The PET/CT images were processed and analyzed for the mouse brains. The uptake (standardized uptake value, SUV) of [¹⁸F]7a was calculated from 15 to 30 minutes post-injection (Figure 5). Our PET imaging data showed: a) [¹⁸F]7a entered the brain with good brain retention: [¹⁸F]7a had a high SUV in mouse brains, with the highest uptake in cerebellar white matter and relatively lower uptake in the neocortex, the distribution of [¹⁸F]7a matched well with the reported S1PR5 expression in CNS that S1PR5 is highly expressed in the oligodendrocytes in the CNS which are found in both gray and white matter but more abundant in white matter regions; b) A-971432 and Ozanimod increased brain uptake by 16.5%, and 30.3%: indicating the uptake of [¹⁸F]7a can be affected by S1PR5 modulators, confirming the in vivo specificity of [¹⁸F]7a toward S1PR5 in CNS; 3) 16g (4 mg/kg) decreased brain uptake by 31.8%: this demonstrated the binding of [¹⁸F]7a can be blocked by its analogue, indicating a reversible binding of [¹⁸F]7a. Pretreatment with three S1PR5 modulators changed the brain uptake, indicating that [¹⁸F]7a has in vivo binding specificity for S1PR5. Notably, both A-971432 and Ozanimod caused an increased uptake of [¹⁸F]7a whereas 16g, the analogue of 7a resulted in a decrease uptake of [¹⁸F]7a. We hypothesize that the increase brain uptake of [¹⁸F]7a is resulted from that both of A-971432 and Ozanimod enhanced influx of [¹⁸F]7a to bind to S1PR5 in the brain because they caused the internalization of S1PR5 in peripheral tissues. In contrast, 16g, the structural similar analogue of 7a, may not cause the internalization and it directly competes with radiotracer [¹⁸F]7a to bind with S1PR5 protein in the brain, reducing the brain uptake of [¹⁸F]7a. Further investigating the interaction of structural different S1PR5 compounds with S1PR5 in vivo may help to explain this disagreement.

Figure 5. — PET/MR/CT imaging of [¹⁸F]7a in C57BL/6 control mice, pretreated with potent S1PR5 modulators **16g**, A-971432, and Ozanimod. (a) Top: Standardized uptake value (SUV) PET/MR images of a control mouse at baseline condition (Top-Left) and mouse pretreated with S1PR5 compound **16g** (4 mg/kg, Top-Right) and S1PR5 agonists A-971432 (4 mg/kg, Bottom-Left) and Ozanimod (4 mg/kg, Bottom-Right). (b) Regional brain uptakes (SUVs) in control mice at baseline (n = 13), with blocking using **16g** (n = 2) and modulation using A-971432 (n = 3) and Ozanimod (n = 3). * Statistically significant with p-value < 0.05, using independent T-tests. *WB = Whole Brain, cWM = Cerebellar White Matter, cGM = Cerebellar Gray Matter, NC = Neocortex, CC = Corpus Callosum, THA = Thalamus, PUT = Putamen, HIP = Hippocampus, PONS = Pons, MID = Midbrain*.

PET/CT imaging in cuprizone-diet mouse model of MS and control mice.

To investigate if PET with [¹⁸F]7a can measure changes in S1PR5 expression associated with demyelination in MS, PET studies were performed in cuprizone-fed mice and control mice. Briefly, 8-week-old female C57BL/6 mice (n = 8, weight: ~19 g) were randomized into a group fed with a standard diet (n=4; control group) or fed with a standard diet mixed with 0.2% (w/w) cuprizone for 4 weeks (n = 4). Cuprizone-induced changes were first assessed by T₂-weighted MRI (week 4). Next, PET imaging of [¹⁸F]7a was performed to investigate S1PR5 expression changes in response to demyelination in the mouse brain. As shown in Figure 6, our PET studies found a decreased whole brain uptake (22.0%) in cuprizone mice. The SUV of whole brain uptake in the cuprizone mice were 0.513, 0.602, and 0.655 at 15, 30, and 60 min respectively versus SUVs of 0.585, 0.700, and 0.757 at 15, 30, and 60 min in control mice (details are presented in Figure S1 of the supplementary information). Within the brain regions, the uptake was decreased in cerebellar white matter (20.0%), cerebellar gray matter (16.7%), neocortex (22.7%), corpus callosum (20.0%), thalamus (~22.2%), putamen (23.9%), hippocampus (20.4%), pons (21.4%), and midbrain (20.3%) (Figure 6b). Together, our PET studies confirmed that [¹⁸F]7a can detect the decreased S1PR5 expression level in the brain of cuprizone-fed mice compared to control mice. This suggests that [¹⁸F]7a has high potential for imaging S1PR5 in the brain for MS.

Figure 6. — PET/MR/CT imaging of [¹⁸F]7a in the cuprizone model of MS (n = 4) and control mice (n = 4). (a) Top: Subject brain CT registered to template MR. Bottom: Standardized uptake value (SUV) PET images on Template MR; (b) Regional brain SUV uptakes in control versus cuprizone mice at 4 weeks. * Statistically significant with p-value < 0.05, using independent T-tests. *WB = Whole Brain, cWM = Cerebellar White Matter, cGM = Cerebellar Gray Matter, NC = Neocortex, CC = Corpus Callosum, THA = Thalamus, PUT = Putamen, HIP = Hippocampus, PONS = Pons, MID = Midbrain*

In vitro autoradiography of [¹⁸F]7a and immunohistology of S1PR5 studies in the cuprizone mouse model of MS.

To confirm if the reduced uptake of [¹⁸F]7a in cuprizone model of MS is associated with decreased expression of S1PR5, we next performed in vitro autoradiography analysis of [¹⁸F]7a and immunofluorescent analysis of S1PR5 in control and cuprizone-fed mouse. Consistent with our PET studies, our autoradiography analysis showed the uptake of [¹⁸F]7a was 30475 ± 1510 PSL/mm² in control mice and 22697 ± 1484 PSL/mm² in cuprizone-fed mice; the uptake of [¹⁸F]7a was 25.5% less in the cuprizone mouse brain compared to control with a p-value of 0.0213 (Figure 7A). To further understand the mechanisms of reduced uptake of [¹⁸F]7a in cuprizone-fed mice, we first characterized the expression of S1PR5 expression using immunofluorescence analysis. Double staining of S1PR5 and oligodendrocyte transcription factor 2 (Olig2) confirmed that S1PR5 is highly expressed in the oligodendrocyte cells, particularly in the white matter regions of the brain such as the corpus callosum and fimbria (Figure 7B). Next, we performed immunofluorescence analysis on the brains of control and cuprizone-fed mice. Immunostaining with myelin basic protein (MBP) confirmed the pathology of cuprizone-fed mice with a dramatic loss of MBP in the corpus callosum (Figure 7C). Furthermore, immunostaining with S1PR5 demonstrated a significant loss of S1PR5 that is associated with MBP loss in the cuprizone mice, particularly in the white matter-rich region such as the corpus callosum (Figure 7C). Quantitative analysis showed 969.7 ± 62.7 S1PR5 positive cells per mm² in the corpus callosum compared to 635.7 ± 30.8 S1PR5 positive cells per mm² in the cuprizone-fed mouse brain, a 34.4% reduction with a p-value of 0.0088 (Figure 7C). Overall, the radioactive distribution of [¹⁸F]7a observed from in vitro autoradiography analysis matched well with our immunohistology analysis using S1PR5 antibody, consistent with our PET imaging data. These results confirmed the specificity of [¹⁸F]7a binding toward S1PR5. The reduced uptake of [¹⁸F]7a resulting from lower expression of S1PR5 was more significant in the white matter-rich regions such as the corpus callosum, in agreement with previous findings that S1PR5 is mainly distributed in the oligodendrocyte cell and indicated a potential oligodendrocytes-driven loss of S1PR5 expression in response to cuprizone treatment. While further characterization of the cellular and molecular mechanism of S1PR5 loss is necessary and will improve our understanding of the pathogenesis of MS, our current data demonstrates the capability of identifying changes in S1PR5 expression in MS using our lead radioligand and provides insights into the spatial information of changes in S1PR5 and its relationship to MS pathology.

Figure 7. — *In vitro* autoradiography of [¹⁸F]7a and immunohistology analysis of the distribution and the expression of S1PR5 in cuprizone-fed mouse brain. (A) Autoradiography study showed a ~25.5% reduced uptake of [¹⁸F]7a in the cuprizone mouse brain compared to sham with a p-value of 0.0213; (B) Immunofluorescent analysis showed S1PR5 is highly expressed in the oligodendrocytes identified by Olig2 staining; (C) Immunofluorescent analysis confirmed the loss of S1PR5 expression that is associated with loss of MBP in cuprizone-fed mice (scale bar = 50 μm).

PET brain imaging studies in nonhuman primates (NHP).

Encouraged by promising results from PET studies in mice, we investigated the brain uptake in a male rhesus macaque (weight ~17.7 kg). We performed 120-minute dynamic PET brain scans twice in the same monkey (~2 weeks apart) with [¹⁸F]7a using an MRS*PET/CT 220 preclinical scanner (MR Solutions, United Kingdom). The PET images were motion-corrected, and a rhesus macaque atlas was normalized to the MR image to extract the regional brain uptakes. [¹⁸F]7a entered the NHP brain quickly and achieved a maximum whole brain uptake with an SUV value of ~1.80 at ~25 min post-injection and slowly washed out from the brain with an SUV value of ~1.66 at 120 min post-injection indicating a very good brain retention and demonstrated clinically favorable tracer kinetics (Fig. 8A). Regional brain analysis showed that [¹⁸F]7a has high uptake in the thalamus, putamen, cerebellar white matter (WM), cerebellar gray matter (GM) and cerebral GM with average max SUV of 1.82, 1.84, 1.58, 2.00 and 1.87 at ~25 min post-injection (Fig. 8B–C) with good reproducibility within the same animal.

Figure 8. — PET brain imaging study of [¹⁸F]7a in macaque. (A) Average whole brain SUV uptake of [¹⁸F]7a in a macaque from two separated scans with > 4 weeks interval. (B) Regional brain SUV uptakes (C) Representative average PET brain image of [¹⁸F]7a (over 120 min) and MR brain image.

Radiometabolite analysis of [¹⁸F]7a in rat and nonhuman primates (NHP).

To check if [¹⁸F]7a formed brain-penetrant radiometabolite(s), we performed HPLC radiometabolite analysis of NHP plasma, rat plasma, and rat brain samples collected post-injection of [¹⁸F]7a (Fig. 9). For the macaque plasma radioactive samples (Fig. 9B&C), at 5 min, only a radioactive peak from parent radiotracer [¹⁸F]7a was detected, indicating ~100% radioactivity is from the parental radiotracer [¹⁸F]7a. For the plasma samples collected at 30, 60, and 90 min post-injection, the major radioactive peak was the parental radiotracer [¹⁸F]7a with retention time T_R = ~15 min, a hydrophilic radiometabolite as peak #A with retention time T_R = ~7.5 min was identified with small percentage (%) of radioactivity as 0%, 13%, 20%, and 25.5% for plasma samples collected at 5, 30, 60, and 90 min post-injection, respectively. Considering the significant retention time difference between this hydrophilic metabolite (T_R = ~7.5 min) and the parental radiotracer [¹⁸F]7a (T_R = ~15 min), this hydrophilic metabolite is unlikely to cross the blood-brain barrier and interfere with the brain uptake of [¹⁸F]7a. Additionally, two negligible radiometabolite peaks (B and C) with retention times of ~9.3 and ~10.2 min were observed with <10% of total radioactivity in the 30, 60, and 90 min plasma samples. These findings confirm the good in vivo metabolic stability of [¹⁸F]7a in macaque, although a small amount of hydrophilic radiometabolite was detected. For the rat plasma and brain tissue samples (Fig. 9A), no detectable radiometabolites were observed, suggesting that [¹⁸F]7a has no radiometabolite that could enter the rat brain and confound with PET measurement. The observed metabolic species difference between NHP and rat does not prevent [¹⁸F]7a as a promising radiotracer for imaging S1PR5 in nonhuman primates or rodents. In addition, after FDA approval of [¹⁸F]7a for human use, further radiometabolite analysis of human plasma samples post-injection is necessary to confirm that [¹⁸F]7a has no radiometabolite that can enter the human brain.

Figure 9. — Radiometabolite analysis of rat plasma and brain samples (A) and nonhuman primate plasma samples (**B&C**) post injection of [¹⁸F]**7a.** HPLC radioactive chromatograms of plasma and brain samples at different time post injection of the radiotracer. The peak with retention time of ~15 minutes is the parental radiotracer.

As stated above, S1PR5 in MS has played a critical role in improving the S1P modulators’ therapeutic efficacy in treating MS. While some of the promising S1P modulators, such as Siponimod and Ozanimod, exhibit dual binding potency for both S1PR1 and S1PR5, the others such as Fingolimod, are potent for multiple S1P receptors. In addition, the mechanisms of S1PR5 in direct contribution to MS remain not fully understood. The expression of S1PR5 in response to disease severity and progression remains unclear. Identification of a specific S1PR5-targeting PET radiotracer that can detect the S1PR5 expression changes in CNS is urgently needed for investigating the S1PR5 pathophysiology roles in animal models or patients with diseases such as MS, Alzheimer’s disease (AD), and Parkinson’s disease (PD). In our current work, we designed and synthesized 27 new compounds through optimization of the naphthalene pharmacophore containing lead S1PR5 compound 7 and further radiosynthesized four C-11 or F-18 labeled radiotracers: [¹¹C]7, [¹⁸F]7a, [¹⁸F]12a, and [¹⁸F]12b. To test the feasibility of imaging S1PR5 in vivo, we performed biodistribution studies in SD rats and discovered that [¹⁸F]7a has the highest brain uptake compared to the other three S1PR5 radiotracers. Consequentially, we performed PET studies in mice and showed that pretreatment with S1P modulators, Ozanimod − a duel S1PR1 and S1PR5 modulator, and A-971432 − a mono-selective S1PR5 modulator, caused an increase in brain uptake compared to controls, when pretreatment with 16g, structurally similar to [¹⁸F]7a caused decrease in brain uptake compared to control mice, suggesting [¹⁸F]7a has binding specificity with S1PR5 in the mouse brain. Cuprizone-induced mouse model of MS is widely used to study the demyelination pathogenesis of MS. Cuprizone diet continually fed for 4 weeks causes demyelination in the brain. Our PET imaging study showed that cuprizone fed mice had decreased brain uptake of [¹⁸F]7a compared to normal control mice, consistent with our in vitro autoradiographic analysis. Furthermore, our S1PR5 immunohistology staining also confirmed the [¹⁸F]7a PET data that cuprizone-fed mice had decreased S1PR5 expression in the brain compared to healthy control mice. More specifically, our immunohistology data demonstrated that the reduced uptake of [¹⁸F]7a and S1PR5 expression is associated with oligodendrocytes cell expression and correlates with demyelination in the cuprizone-fed mice model of MS. Together, through our efforts on design, synthesis and radiosynthesis, and in vivo and in vitro characterization in rodent and nonhuman primates, we identified a promising specific F-18 labeled radiotracer [¹⁸F]7a for imaging S1PR5 in vivo for MS. Further dosimetry and toxicity validation is warranty prior to seeking USA FDA approval for human use.

CONCLUSION

In summary, we designed and synthesized a total of 27 new S1PR5 ligands and the reference compound 7, nine of these compounds displayed high potency and selectivity for S1PR5. We successfully radiosynthesized four C-11 or F-18 labeled radioligands [¹¹C]7, [¹⁸F]7a, [¹¹C]12a, and [¹⁸F]12b with moderate to good radiochemical yield, high radiochemical purity, and high molar activity. Our biodistribution study showed that [¹⁸F]7a has the highest brain uptake in Sprague Dawley rats compared to the other three radiotracers. Our direct saturation binding assay and saturation autoradiography studies showed that [¹⁸F]7a is very potent for S1PR5 with K_d values < 10 nM for recombinant human S1PR5 cell membrane, C57BL/6 mouse brain tissues, and 27.6 ± 3.6 nM for the human cerebral cortex respectively. Our PET studies in mice at control condition and pretreatment with S1PR5 modulators demonstrated that [¹⁸F]7a had specific binding with S1PR5 in the brain. PET studies in cuprizone mouse model of MS demonstrated [¹⁸F]7a can detect the loss of oligodendrocytes S1PR5 expression in cuprizone fed mice that correlates with demyelination. In addition, our HPLC radiometabolite analysis in rodents and nonhuman primates indicated no radiometabolite enters the rat brain to confound PET measurements. Together, our data suggested that [¹⁸F]7a is a promising radiotracer for imaging S1PR5 in CNS diseases.

EXPERIMENTAL SECTION

All compounds purity is determined by using a Thermo Fisher Scientific Vanquish^™ VF-D40-A analytic HPLC system equipped with an Phenomenex Prodigy ODS-3 column (5 μm, 100 Å, LC Column 250 × 4.6 mm). A different gradient (30–98%v/v) of methanol in 0.1 M ammonium formate aqueous solution that contained 0.1 %v/v formic acid was used as eluent (flow rate: 1 mL/min; wavelength monitored: 254 nm). All tested compounds are >95% pure determined by analytic HPLC system.

Animal Studies.

All animal experiments were performed following the protocols evaluated and approved by the Institutional Animal Care and Use Committee (IACUC) of Washington University School of Medicine in St. Louis, Missouri, USA, (Ethics Approval Number: 20–0101 for rodent studies and 21–0131 for nonhuman primate (NHP) studies ) and were conducted in accordance with the National Research Council’s Guidelines for the Care and Use of Laboratory Animals. NHP research was performed in a dedicated NHP PET imaging suite using adult male macaques (Tulane National Primate Research Center, Covington, LA) weighing ~10 kg. PET imaging for NHPs followed established protocols.^{40, 41} Before transport to the scanner suite, macaques were anesthetized with ketamine (10–20 mg/kg) and administered glycopyrrolate (0.013–0.017 mg/kg) to minimize saliva secretion. Anesthesia was maintained during imaging with 1.0–1.5% isoflurane in a mixture of 40% N₂O and 60% O₂. Radiotracers and fluids were delivered via a percutaneous venous catheter, and a femoral artery catheter was used for arterial blood sampling. Male Sprague-Dawley rats (6–7 weeks old, 200–320 g; Charles River, Wilmington, MA) were used for biodistribution studies, following previously reported methods.⁴¹ Female C57BL/6J mice (strain 000664) were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and kept in normal day–night cycle (12/12) with ad libitum access to food and water. At eight weeks old, mice were fed a 0.2% cuprizone (Sigma-Aldrich) diet for four weeks to induce cerebral demyelination and inflammation, after which they were used for PET imaging analysis. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Washington University in St. Louis.

General Section:

All reagents and chemicals were purchased commercially and used as received, unless otherwise stated. Reactions were monitored by thin-layer chromatography (TLC) using silica gel 60 F₂₅₄ (EMD Chemicals Inc., Billerica, MA) with compounds visualized under UV light (λ = 254 nm). Flash column chromatography was conducted using 230–400 mesh silica gel (SiliCycle Inc., Quebec, Canada). All deuterated solvents were purchased from Cambridge Isotope Laboratories. ¹H and ¹³C NMR spectra were recorded on a 400 MHz Varian instrument. Chemical shifts were reported in parts per million (ppm) and were calibrated using a residual undeuterated solvent as an internal reference (CDCl₃: δ 7.26 ppm; CD₃OD: δ 3.31 ppm; DMSO-d₆: δ 2.50 ppm). Coupling constants (J) are reported in Hertz (Hz). Multiplicities are indicated by s (singlet), d (doublet), t (triplet), q (quartet), p (pentet), h (hextet), m (multiplet) and br (broad). High-resolution positive ion mass was acquired by a Bruker MaXis 4G Q-TOF mass spectrometer with an electrospray ionization source. The C18 Sep-Pak light cartridges (Inc. Catalog # WAT023501), and C18 Sep-Pak Plus cartridges (Inc. Catalog # WAT020515) were purchased from Waters Corporation, Milford, MA.

General procedure A: Synthesis of compound 3.

A mixture of 4-bromonaphthalen-2-ol (0.30 g, 1.34 mmol), cis-4-(tert-butyl)cyclohexyl methanesulfonate (0.63 g, 2.68 mmol) and cesium carbonate (1.27 g, 3.88 mmol) was dissolved in a solvent mixture of t-BuOH (5 mL) and 2-butanone (3.5 mL) and heated at 80 °C overnight. After cooling to room temperature, the reaction mixture was quenched with water and ether. The mixture was extracted with ether (10 mL × 3), and the combined organic layers were dried over anhydrous MgSO₄, filtered, and concentrated. The crude was purified by column chromatography (Hexane/Ethyl Acetate: 20/1) to afford the compound 3.

General procedure B: Synthesis of compound 4.

Dimethylzinc (2M in toluene; 2 equiv, 0.72 mL) was added under nitrogen to 1-bromo-3-(((trans)-4-(tert-butyl)cyclohexyl)-oxy)naphthalene 3 (0.71 mmol) and Pd(dppf)Cl₂ (0.3 mmol) in dioxane (7 mL) and the resulting pale yellow solution was heated at reflux for 2 h. Upon cooling, the mixture was quenched by the addition of methanol, and then diluted with ethyl ether, washed with 1M hydrochloric acid and water and dried. The organic phase was dried over NaSO₄, filtered and concentrated. The crude was purified by column chromatography (Hexane/Ethyl Acetate: 15/1) to afford the product 4.

General procedure C: Synthesis of compound 4a.

1-Bromo-3-(((trans)-4-(tert-butyl)cyclohexyl)-oxy)naphthalene 3 (0.3111 g, 0.861 mmol) was dissolved in THF (10 mL) and cooled to −78 °C under nitrogen atmosphere. After stirring for 10 minutes, n-BuLi (0.42 mL, 1.03 mmol, 2.5 M in hexanes) was added dropwise. The reaction mixture was stirred at −78 °C for 30 min to ensure complete halogen exchange. Subsequently, NFSI (312.5 mg, 0.99 mmol) in THF (2 mL) was added dropwise to the stirring mixture. After the addition was completed, the reaction was stirred for another 2 hours at −78 °C and then warmed to room temperature overnight. The reaction mixture was quenched with water (10 mL) and the aqueous phase was extracted with ethyl acetate (3 × 50 mL). The resulting organic phase was washed with brine, dried over sodium sulfate, filtered and the solvents removed under reduced pressure. The crude product is purified by column chromatography (Hexane /Ethyl Acetate: 18/1) on silica gel to give the desired product.

General procedure D: Synthesis of compound 4b.

i): The appropriate brominated ethyl 1-bromo-3-(((trans)-4-(tert-butyl)cyclohexyl)oxy)naphthalene (0.07 g, 0.194 mmol, 1.0 equiv) and bis(pinacolato)diboron (0.0594 g, 0.234 mmol, 1.2 equiv) were suspended in 3.50 mL of 1,4-dioxane under a nitrogen atmosphere. Pd(dppf)Cl₂ (7 %) and KOAc (2.0 equiv) were then added, and the mixture was heated to 105 °C for 10 h. After being cooled down to room temperature, the solution was added water and ethyl acetate, and the reaction mixture was extracted with ethyl acetate, washed with water, saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. ii): The crude products were purified by column chromatography (Hexane/Ethyl Acetate: 5/1) to give the aryl boron ester. Subsequently, NaBO₃·4H₂O (1.5 equiv, 1.769 mmol, 0.272 g) was added to a stirred suspension of the boron eater (0.98 mmol, 0.40 g) in THF (7 mL) and water (7 mL) and the reaction was stirred at room temperature for 2 h. The reaction was quenched by NH₄Cl (aq) and extracted with ethyl acetate, dried by sodium sulfate, filtered and concentrated. iii): The crude was purified by column chromatography (Hexane/Ethyl Acetate: 15/1) to afford the naphthol product. Finally, A 25 mL round bottom flask was charged with 3-(((trans)-4-(tert-butyl)cyclohexyl)oxy)naphthalen-1-ol (0.432 mmol, 0.129 g), K₂CO₃ (2 equiv, 0.86 mmol, 0.12 g) and DMF (3 mL). CH₃I (0.91 mmol, 0.13 g) was added, and reaction was stirred overnight at room temperature. The reaction was quenched with water, extracted with ethyl acetate, washed by water, brine, dried over Na₂SO₄, filtered and concentrated. The crude was purified by column chromatography (Hexane/Ethyl Acetate: 25/1) to afford the desired product.

General procedure E: Synthesis of compound 5, 5a and 5b.

To a solution of 4, 4a-4b (0.453 mmol, 1.0 equiv) in dry dichloromethane (4 mL) was added SnCl₄ (0.63 mL, 0.61 mmol, 1.35 equiv) dropwise at 0 °C and the mixture was stirred at 0 °C for 1 h. Dichloromethyl methyl ether (0.07 mL, 0.61 mmol, 1.35 equiv) was added dropwise at 0 °C, and the mixture was stirred at 0 °C for 1 h before warming to room temperature and stirring for an additional 1 h. The reaction was quenched by the addition of ice-water and the mixture was stirred at room temperature for 1 h. The mixture was extracted with dichloromethane (3 × 20 mL). The combined organic layers were washed with saturated brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product is purified by column chromatography (Hexane/Ethyl Acetate: 10/1) to afford the products 5, 5a and 5b.

General procedure F: Synthesis of compound 6, 6a and 6b.

Ethyl isonipecotate (0.05 mL, 0.209 mmol, 1.0 equiv) was added at room temperature to a solution of 5, 5a-5b (0.209 mmol, 1.0 equiv) in anhydrous ethyl acetate (2.2 mL). The mixture was stirred at 45 °C for 4 h. The mixture was cooled to room temperature and sodium triacetoxyborohydride (0.066 g, 0.32 mmol, 1.5 equiv) was added in portions. The mixture was stirred at room temperature overnight. The reaction mixture was diluted with ethyl acetate (10 mL), washed with water (10 mL), saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product is purified by column chromatography (Hexane/Ethyl Acetate: 4/1) on silica gel to give the products.

General procedure G: Synthesis of compound 7, 7a and 7b.

To a solution of compound 6, 6a-6b (0.0699 mmol) in THF/MeOH/H₂O (1 mL /1 mL/0.5 mL) was added lithium hydroxide monohydrate at room temperature, the reaction mixture was stirred at rt for 2 h. The mixture was diluted with water and adjusted to pH < 3 with 1N HCl (aq), extracted with ethyl acetate (10 mL), washed with saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product is purified by column chromatography (MeOH/Ethyl Acetate: 2/1) on silica gel to give the products.

General procedure H: Synthesis of compound 9.

To a solution of compound 8 (0.453 mmol, 1.0 equiv) in dry dichloromethane (4 mL), SnCl₄ (0.63 mL, 0.61 mmol, 1.35 equiv) was added dropwise at 0 °C and the reaction mixture was stirred at 0 °C for 1 h. Dichloromethyl methyl ether (0.07 mL, 0.61 mmol, 1.35 equiv) was then added dropwise at 0 °C, followed by stirring at 0 °C for 1 hour and warming to room temperature for an additional 1h. The reaction was quenched by the addition of ice water and the mixture was stirred at room temperature for 1 h. The mixture was extracted with dichloromethane (3 × 20 mL). The combined organic layers were washed with saturated brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product is purified by column chromatography (Hexane/Ethyl Acetate: 10/1) to afford the product. Then, the previous step product (0.194 mmol, 1.0 equiv), Pd(dppf)Cl₂ (7 %), KOAc (2.0 equiv), and bis(pinacolato)diboron (0.234 mmol, 1.2 equiv) were suspended in 3.50 mL of 1,4-dioxane under a nitrogen atmosphere. And the mixture was heated to 95 °C for 10 h. After being cooled down to room temperature, the solution was added water and ethyl acetate, and the reaction mixture was extracted with ethyl acetate, washed with water, saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude products were purified by column chromatography (Hexane/Ethyl Acetate: 5/1) to give the compound 9.

General procedure I: Synthesis of compound 10.

NaBO₃·4H₂O (1.5 equiv, 1.769 mmol) was added to a stirred suspension of the boron eater 9 (1.0 equiv, 0.98 mmol) in THF (5 mL) and water (5 mL) and the reaction was stirred at room temperature for 2 h. The reaction was quenched by NH₄Cl (aq) and extracted with ethyl acetate, dried by Na₂SO₄, filtered and concentrated. The crude was purified by column chromatography (Hexane/Ethyl Acetate: 15/1) to afford the product.

General procedure J: Synthesis of compound 11a.

To a stirred solution of 7-(((trans)-4-(tert-butyl)cyclohexyl)oxy)-2-hydroxy-5-methyl-1-naphthaldehyde 10 (0.3 mmol) and Methyl iodine (0.6 mmol) in DMF (10 mL) at room temperature was added K₂CO₃ (0.63 mmol). The reaction was stirred at room temperature overnight. Upon completion, the mixture was quenched with ethyl acetate and water, The mixture was extracted with ethyl acetate (3 × 20 mL). The combined organic layers were washed with saturated brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude was purified by column chromatography on silica gel (Hexane/Ethyl Acetate: 40/1) to give product 11a.

General procedure K: Synthesis of compound 11b.

To a stirred solution of 7-(((trans)-4-(tert-butyl)cyclohexyl)oxy)-2-hydroxy-5-methyl-1-naphthaldehyde 10 (0.3 mmol) and 2-fluoroethyl 4-methylbenzenesulfonate (0.6 mmol) in CH₃CN (10 mL) at room temperature was added K₂CO₃ (0.63 mmol). The reaction was heated to 80 °C and stirred overnight. Upon completion, the mixture was cooled to room temperature and moved all the solvents. The residue was dissolved in 40 ml DCM, and the mixture was filtered and then the DCM was removed under reduced pressure. The crude was purified by column chromatography on silica gel (Hexane/Ethyl Acetate: 40/1) to give product 11b.

General procedure L: Synthesis of compound 12a and 12b.

Ethyl isonipecotate (0.05 mL, 0.209 mmol, 1.0 equiv) was added at room temperature to a solution of 11 (0.209 mmol, 1.0 equiv) in anhydrous ethyl acetate (2.2 mL). The mixture was stirred at 60 °C for 3 h. The mixture was cooled to room temperature and sodium triacetoxyborohydride (0.066 g, 0.32 mmol, 1.5 equiv) was added in portions. The mixture was stirred at 60 °C overnight. The reaction mixture was diluted with ethyl acetate (10 mL), washed with water (10 mL), saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product is purified by column chromatography (Hexane/Ethyl Acetate: 4/1) on silica gel to give the products. The product from the previous step (0.0699 mmol) was dissolved in a mixture of THF/MeOH/H₂O (1 mL /1 mL/0.5 mL), and lithium hydroxide monohydrate was added at room temperature, the reaction mixture was stirred at rt for 2 h. The mixture was diluted with water and adjusted to pH < 3 with 1N HCl (aq), extracted with ethyl acetate (10 mL), washed with saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product is purified by column chromatography (MeOH/Ethyl Acetate: 2/1) on silica gel to give the product 12.

General procedure M: Synthesis of compound 15.

Ethyl isonipecotate (0.05 mL, 0.209 mmol, 1.0 equiv) was added at room temperature to a solution of 14 (0.209 mmol, 1.0 equiv) in methanol (2.2 mL). The mixture was stirred at 70 °C for 3 h. The mixture was cooled to room temperature and NaBH₃CN (0.32 mmol, 1.5 equiv) was added in portions. The mixture was stirred at 70 °C overnight. The reaction mixture was diluted with ethyl acetate (10 mL), washed with water (10 mL), saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product is purified by column chromatography (Hexane/Ethyl Acetate: 4/1) on silica gel to give the product 15.

General procedure N: Synthesis of compound 16a-b.

A mixture of compound 5 (0.3 mmol, 1.0 equiv), amino acid salts (0.45 mmol, 1.5 equiv), and DIPEA (0.40 mmol, 1.3 equiv) in DCE (5 mL) was stirred at room temperature for 1 h. Subsequently, NaBH(OAc)₃ (0.51 mmol, 1.77 equiv) was added, and the reaction was stirred at room temperature overnight. The resulting mixture was then diluted with water (5 mL) and extracted with DCM (5 mL x 3). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na₂SO₄ and filtered. The solvent was evaporated under reduced pressure to give the residue, which was purified by column chromatography on silica gel (petroleum ether/EtOAc) to yield the product. The intermediate product was dissolved in a THF/H₂O mixture (1.4 mL/0.6 mL), and lithium hydroxide was added at room temperature. The reaction mixture was stirred overnight at room temperature. Upon completion, the mixture was diluted with water and adjusted to pH < 6 using 1N HCl (aq). The solution was extracted with ethyl acetate (10 mL), washed with saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product is purified by column chromatography (MeOH/Ethyl Acetate: 2/1) on silica gel to give the product 16a-b.

General procedure O: Synthesis of compound 16c.

A solution of 5 (0.3 mmol, 1.0 eq), 3-amino-2-fluoropropanoic acid hydrochloric acid salts (0.45 mmol, 1.5 eq), CH3COOH (2 drops), and DIPEA (0.40 mmol, 1.3 eq) in EtOH (6 mL) was stirred at 80 °C for 2 h. The reaction mixture was cooled to rt and NaBH₃CN (0.51 mmol, 1.77 equiv) was added, and the reaction was stirred at 80 °C overnight. The resulting mixture was then diluted with water (5 mL) and extracted with ethyl estate (5 mL x 3). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na₂SO₄ and filtered. The solvent was evaporated under reduced pressure to give the residue, which was purified by column chromatography on silica gel (petroleum ether /EtOAc) to yield the product 16c.

General procedure P: Synthesis of compound 16d-f.

i): A solution of 5 (0.3 mmol, 1.0 eq), amino ester hydrochloric acid salts (0.45 mmol, 1.5 eq), and DIPEA (0.40 mmol, 1.3 eq) in DCE (5 mL) was stirred at rt for 1 h. The reaction mixture was cooled to rt and NaBH(OAc)₃ (0.51 mmol, 1.77 equiv) was added, and the reaction was stirred at rt overnight. The resulting mixture was then diluted with water (5 mL) and extracted with ethyl estate (5 mL x 3). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na₂SO₄ and filtered. The solvent was evaporated under reduced pressure to give the residue, which was purified by column chromatography on silica gel (petroleum ether /EtOAc) to yield the product. ii): The intermediate product (0.1 mmol) was dissolved in MeOH (2 mL), followed by the addition of CH₃COOH (2 drops) and formaldehyde solution (36.5–38% in H₂O, 1.0 mmol) at room temperature. The reaction mixture was stirred at room temperature for 1 hour, after which NaBH₃CN (0.2 mmol) was added, and the reaction was stirred overnight at room temperature. Upon completion, the mixture was diluted with water and extracted with ethyl acetate (10 mL). The combined organic layers were washed with saturated brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (Hexane/Ethyl Acetate: 4/1) on silica gel to yield the desired product. iii): The product was then dissolved in a THF/MeOH/H₂O mixture (1 mL/1 mL/0.5 mL), and lithium hydroxide monohydrate was added at room temperature, the reaction mixture was stirred at rt for 2 h. Upon completion, the mixture was diluted with water and adjusted to pH < 6 using 1N HCl (aq). The solution was extracted with ethyl acetate (10 mL), washed with saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product is purified by column chromatography (MeOH/Ethyl Acetate: 2/1) on silica gel to give the products.

General procedure Q: Synthesis of compound 16g-i & m.

A solution of 5 (0.3 mmol, 1.0 equiv) and primary amine materials (0.39 mmol, 1.3 equiv) in DCE (5 mL) was stirred at rt for 1 h. Then the reaction mixture was added NaBH(OAc)₃ (0.54 mmol, 1.8 equiv) at rt, and the reaction was stirred at rt overnight. The resulting mixture was then diluted with water (5 mL) and extracted with ethyl estate (5 mL × 3). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na₂SO₄ and filtered. The solvent was evaporated under reduced pressure to give the residue, which was purified by column chromatography on silica gel (MeOH/Ethyl Acetate: 2/1) to yield the products.

General procedure R: Synthesis of compound 16j.

A solution of 5 (0.3 mmol, 1.0 eq) and amino acid (0.45 mmol, 1.5 eq), in EtOH (6 mL) was stirred at 80 °C for 2 h. The reaction mixture was cooled to rt and NaBH₃CN (0.51 mmol, 1.77 equiv) was added, and the reaction was stirred at 80 °C overnight. The resulting mixture was then diluted with water (5 mL) and extracted with ethyl estate (5 mL x 3). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na₂SO₄ and filtered. The solvent was evaporated under reduced pressure to give the residue, which was purified by column chromatography on silica gel (EtOAc /MeOH) to yield the product 16j.

General procedure S: Synthesis of compound 16k-l, 16n and 18a-f.

A solution of 5 or 17a-f (0.3 mmol, 1.0 equiv), amine hydrochloric acid salts (0.45 mmol, 1.5 equiv) and DIPEA (0.40 mmol, 1.3 equiv) in DCE (5 mL) was stirred at rt for 1 h. Then the reaction mixture was added NaBH(OAc)₃ (0.51 mmol, 1.77 equiv) at rt, and the reaction was stirred at rt overnight. The resulting mixture was then diluted with water (5 mL) and extracted with ethyl estate (5 mL x 3). The combined organic layers were washed with brine (10 mL), dried over anhydrous Na₂SO₄ and filtered. The solvent was evaporated under reduced pressure to give the residue, which was purified by column chromatography on silica gel (MeOH/Ethyl Acetate: 2/1) to yield the products.

General procedure T: Synthesis of compound 16o and 16p.

Ethyl isonipecotate (0.15 mmol, 1.0 equiv) and DIPEA (0.13 mmol, 1.3 equiv) was added at room temperature to a solution of 5 (0.10 mmol, 1.0 equiv) in anhydrous DCE (2.2 mL). The mixture was stirred at 45 °C for 3 h. The mixture was cooled to room temperature and sodium triacetoxyborohydride (0.17 mmol, 1.7 equiv) was added in portions. The mixture was stirred at rt overnight. The reaction mixture was diluted with ethyl acetate (10 mL), washed with water (10 mL), saturated brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The crude product is purified by column chromatography (Hexane/Ethyl Acetate: 4/1) on silica gel to give the product I. The product I (0.1 mmol) was dissolved in DCM (3 mL) and cooled to 0 °C. TFA was added dropwise at 0 °C, and the reaction was stirred at room temperature for 40 minutes. The reaction mixture was quenched with K₂CO₃, extracted with DCM, and the combined organic layers were washed with brine (10 mL), dried over anhydrous Na₂SO₄, and filtered. The solvent was evaporated under reduced pressure to give the residue, which was purified by column chromatography on silica gel (MeOH/Ethyl Acetate: 2/1) to yield the product II. The product II (0.1 mmol) was dissolved in MeOH (2 mL), followed by the addition of CH₃COOH (2 drops) and an aqueous formaldehyde solution (1.0 mmol) at room temperature. The reaction mixture was stirred at room temperature for 1 hour, after which NaBH₃CN (0.2 mmol) was added, and the reaction was stirred overnight at room temperature. Upon completion, the mixture was diluted with water and extracted with ethyl acetate (10 mL). The combined organic layers were washed with saturated brine, dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography (Hexane/Ethyl Acetate: 1/1) on silica gel to yield the desired product 16o.

The product II (0.1 mmol) and 2-fluoroethyl 4-methylbenzenesulfonate (0.2 mmol) were dissolved in CH₃CN (10 mL) at room temperature. K₂CO₃ (0.21 mmol) was added, and the mixture was heated to 80 °C and stirred overnight. Upon completion, the mixture was cooled to room temperature and moved all the solvents. The residue was dissolved in 40 ml DCM, and the mixture was filtered and then the DCM was removed under reduced pressure. The crude was purified by column chromatography on silica gel (Hexane/Ethyl Acetate: 1/1) to give product 16p.

1-Bromo-3-trans-4-(tert-butyl)cyclohexyl)oxy)naphthalene (3). Synthesized by general procedure A from commercial available 2.White solid; yield: 43%; MP: 117−119 °C; ¹H NMR (400 MHz, Chloroform-d) δ 8.13 – 8.10 (m, 1H), 7.70 – 7.67 (m, 1H), 7.49 (d, J = 2.4 Hz, 1H), 7.48 – 7.40 (m, 2H), 7.16 – 7.12 (m, 1H), 4.25 (tt, J = 10.9, 4.3 Hz, 1H), 2.29 – 2.23 (m, 2H), 1.92 – 1.88 (m, 2H), 1.49 – 1.39 (m, 2H), 1.21 – 1.12 (m, 3H), 0.90 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 155.3, 135.1, 127.5, 127.0, 127.0, 126.9, 124.8, 123.7, 123.3, 108.7, 77.2, 47.2, 32.4, 32.3, 27.6, 25.5.

3-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-1-methylnaphthalene (4). Synthesized by general procedure B from 3. White solid; yield: 89%; MP: 110−112 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.93 (d, J = 8.3 Hz, 1H), 7.75 (d, J = 8.1 Hz, 1H), 7.47 (t, J = 7.4 Hz, 1H), 7.39 (t, J = 7.5 Hz, 1H), 7.07 – 7.05 (m, 2H), 4.35 – 4.27 (m, 1H), 2.68 (s, 3H), 2.35 – 2.31 (m, 2H), 1.95 – 1.91 (m, 2H), 1.53 – 1.43 (m, 2H), 1.28 – 1.10 (m, 3H), 0.94 (s, 9H). ¹³C NMR (100 MHz, Chloroform-d) δ 155.18, 136.15, 134.86, 128.18, 127.23, 125.99, 123.93, 123.24, 120.48, 106.46, 76.36, 47.33, 32.35, 27.65, 27.44, 25.58, 19.25.

3-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-1-fluoronaphthalene (4a). Synthesized by general procedure C from 3. White solid; yield: 83%; MP: 121−124 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.98 (d, J = 8.3 Hz, 1H), 7.70 (d, J = 8.3 Hz, 1H), 7.48 – 7.44 (m, 1H), 7.38 – 7.34 (m, 1H), 6.96 (d, J = 2.1 Hz, 1H), 6.85 (dd, J = 12.0, 2.2 Hz, 1H), 4.26 (tt, J = 10.9, 4.3 Hz, 1H), 2.29 – 2.25 (m, 2H), 1.92 – 1.88 (m, 2H), 1.49 – 1.46 (m, 1H), 1.43 – 1.39 (m, 1H), 1.24 – 1.10 (m, 3H), 0.90 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 159.4 (d, J_C-F = 251.0 Hz), 155.6 (d, J_C-F = 12.0 Hz), 135.1 (d, J_C-F = 6.0 Hz), 127.3, 126.3, 123.6, 120.5 (d, J_C-F = 5.0 Hz), 119.2 (d, J_C-F = 12.0 Hz), 104.4, 103.8 (d, J_C-F = 22.0 Hz), 77.1, 47.3, 32.4, 32.2, 27.6, 25.5.

3-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-1-methoxynaphthalene (4b). Synthesized by general procedure D from 3. Yellow solid; yield: 33%; MP: 83−85 °C; ¹H NMR (400 MHz, Chloroform-d) δ 8.17 (d, J = 8.3 Hz, 1H), 7.68 (d, J = 8.2 Hz, 1H), 7.47 – 7.43 (m, 1H), 7.34 – 7.30 (m, 1H), 6.78 (s, 1H), 6.53 (s, 1H), 4.35 – 4.27 (m, 1H), 3.98 (s, 3H), 2.36 – 2.32 (m, 2H), 1.94 – 1.90 (m, 2H), 1.53 – 1.43 (m, 2H), 1.24 – 1.09 (m, 3H), 0.94 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 156.6, 156.1, 135.1, 126.9, 126.2, 122.7, 121.9, 121.4, 100.0, 98.7, 76.4, 55.5, 47.3, 32.3, 27.6, 27.6, 25.6.

2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-methyl-1-naphthaldehyde (5). Synthesized by general procedure E from 4. White solid; yield: 71%; MP: 91−94 °C; ¹H NMR (400 MHz, Chloroform-d) δ 10.85 (s, 1H), 9.37 (d, J = 8.7 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.61 (t, J = 7.7 Hz, 1H), 7.44 (t, J = 7.6 Hz, 1H), 7.13 (s, 1H), 4.42 – 4.35 (m, 1H), 2.73 (s, 3H), 2.26 – 2.22 (m, 2H), 1.92 – 1.88 (m, 2H), 1.57 – 1.47 (m, 2H), 1.22 – 1.18 (m, 3H), 0.89 (s, 9H). ¹³C NMR (100 MHz, Chloroform-d) δ 192.07, 162.87, 145.05, 131.85, 129.24, 127.80, 125.43, 124.62, 123.96, 116.87, 116.52, 79.03, 47.05, 32.65, 32.30, 27.58, 25.44, 20.78. HRMS (ESI): m/z calculated for C₂₂H₂₈O₂Na [M + Na]⁺: 347.1982. Found: 347.1977.

2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-fluoro-1-naphthaldehyde (5a). Synthesized by general procedure E from 4a. White solid; yield: 82%; MP: 101−103 °C; ¹H NMR (400 MHz, Chloroform-d) δ 10.78 (s, 1H), 9.35 – 9.32 (m, 1H), 8.02 (d, J = 8.3 Hz, 1H), 7.68 – 7.64 (m, 1H), 7.48 – 7.44 (m, 1H), 6.98 (d, J = 12.1 Hz, 1H), 4.32 (tt, J = 10.9, 4.4 Hz, 1H), 2.27 – 2.22 (m, 2H), 1.93 – 1.89 (m, 2H), 1.59 – 1.49 (m, 2H), 1.22 – 1.09 (m, 3H), 0.89 (s, 9H).¹³C NMR (100 MHz, Chloroform-d) δ 191.0, 164.1 (d, J_C-F = 11.0 Hz), 163.9 (d, J_C-F = 262.0 Hz), 133.1 (d_J-_F = 7.0 Hz), 130.8, 125.0, 125.0, 120.6, 120.6 (d, J_C-F = 6.0 Hz), 119.0 (d_J-_F = 11.0 Hz), 115.0, 99.9 (d_J-_F = 24.0 Hz), 79.5, 47.0, 32.5, 32.3, 27.6, 25.4. HRMS (ESI) m/z [M+Na]⁺ calcd for C₂₁H₂₅FNaO₂: 351.1731, found: 351.1728.

2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-methoxy-1-naphthaldehyde (5b). Synthesized by general procedure E from 4b. White solid; yield: 54%; MP: 80−82 °C; ¹H NMR (400 MHz, Chloroform-d) δ 10.73 (s, 1H), 9.37 (d, J = 8.7 Hz, 1H), 8.18 (dd, J = 8.3, 1.4 Hz, 1H), 7.63 – 7.59 (m, 1H), 7.42 – 7.37 (m, 1H), 6.55 (s, 1H), 4.38 – 4.31 (m, 1H), 4.07 (s, 3H), 2.27 – 2.22 (m, 2H), 1.93 – 1.89 (m, 2H), 1.58 – 1.49 (m, 2H), 1.20 – 1.09 (m, 3H), 0.89 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 190.8, 165.5, 162.4, 132.7, 130.2, 125.0, 124.3, 121.9, 94.4, 79.6, 55.9, 47.1, 32.6, 32.3, 27.6, 25.5.

Ethyl 1-((2-(((trans)-4-(tert-butyl)cyclohexyl)oxy)-4-methylnaphthalen-1-yl)methyl)piperidine-4-carboxylate (6). Synthesized by general procedure F from 5. Colorless oil; yield: 50%; ¹H NMR (400 MHz, Chloroform-d) δ 8.26 (d, J = 8.6 Hz, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.50 – 7.46 (m, 1H), 7.41 – 7.37 (m, 1H), 7.10 (s, 1H), 4.24 – 4.18 (m, 1H), 4.12 (q, J = 7.2 Hz, 2H), 3.91 (s, 2H), 2.94 (dd, J = 11.3, 4.1 Hz, 2H), 2.69 (s, 3H), 2.31 – 2.12 (m, 5H), 1.90 – 1.81 (m, 4H), 1.71 (td, J = 12.8, 12.2, 3.6 Hz, 2H), 1.47 (q, J = 11.6 Hz, 2H), 1.24 (t, J = 7.1 Hz, 3H), 1.16 – 1.09 (m, 3H), 0.90 (s, 9H). ¹³C NMR (100 MHz, Chloroform-d) δ 175.5, 153.4, 135.2, 134.6, 128.5, 125.8, 125.5, 124.0, 123.3, 119.1, 117.4, 78.3, 60.1, 52.9, 52.1, 47.3, 41.5, 33.0, 32.3, 28.4, 27.6, 25.6, 19.8, 14.2. HRMS (ESI): m/z calculated for C₃₀H₄₄NO₃ [M + H]⁺: 466.3316. Found: 466.3317.

Ethyl 1-((2-(((trans)-4-(tert-butyl) cyclohexyl) oxy)-4-fluoronaphthalen-1-yl) methyl) piperidine-4-carboxylate (6a). Synthesized by general procedure F from 5a. Yellow solid; yield: 53%; MP: 80−82 °C; ¹H NMR (400 MHz, Chloroform-d) δ 8.21 (d, J = 8.6 Hz, 1H), 8.00 (d, J = 8.3 Hz, 1H), 7.53 – 7.48 (m, 1H), 7.38 (t, J = 7.6 Hz, 1H), 6.96 (d, J = 12.4 Hz, 1H), 4.19 – 4.09 (m, 3H), 3.86 (s, 2H), 2.94 – 2.89 (m, 2H), 2.32 – 2.25 (m, 1H), 2.22 – 2.12 (m, 4H), 1.90 – 1.81 (m, 4H), 1.74 – 1.64 (m, 2H), 1.51 – 1.42 (m, 2H), 1.24 (t, J = 7.1 Hz, 3H), 1.16 – 1.08 (m, 3H), 0.89 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 175.4, 158.8 (d, J_C-F = 250.0 Hz), 153.4 (d, J_C-F = 10.0 Hz), 135.0 (d, J_C-F = 12.0 Hz), 127.0, 124.9, 123.6, 120.4 (d, J_C-F = 6.0 Hz), 119.3 (d, J_C-F = 15.0 Hz), 116.7, 100.4 (d, J_C-F = 22.0 Hz), 78.3, 60.1, 52.8, 51.9, 47.2, 41.5, 32.8, 32.3, 28.3, 27.6, 25.5, 14.2; ¹⁹F NMR (375 MHz, Chloroform-d) δ −121.8. HRMS (ESI) m/z [M+H]⁺ calcd for C₂₉H₄₁FNO₃: 470.3065, found: 470.3068.

Ethyl 1-((2-(((trans)-4-(tert-butyl) cyclohexyl) oxy)-4-methoxynaphthalen-1-yl) methyl) piperidine-4-carboxylate (6b). Synthesized by general procedure F from 5b. Yellow oil; yield: 51%; ¹H NMR (400 MHz, Chloroform-d) δ 8.17 – 8.15 (m, 2H), 7.49 – 7.45 (m, 1H), 7.35 – 7.31 (m, 1H), 6.59 (s, 1H), 4.18 – 4.14 (m, 1H), 4.10 (q, J = 7.0 Hz, 2H), 3.99 (s, 3H), 3.83 (s, 2H), 2.94 – 2.90 (m, 2H), 2.30 – 2.09 (m, 5H), 1.88 – 1.79 (m, 4H), 1.72 – 1.62 (m, 2H), 1.48 – 1.42 (m, 2H), 1.22 (t, J = 7.1 Hz, 4H), 1.13 – 1.07 (m, 3H), 0.88 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 175.6, 157.0, 154.1, 134.8, 126.7, 124.8, 123.0, 121.9, 121.8, 98.7, 96.3, 79.0, 60.1, 55.5, 52.8, 52.1, 47.3, 41.6, 33.0, 32.3, 28.4, 27.6, 25.6, 14.2. HRMS (ESI) m/z [M+H]⁺ calcd for C₃₀H₄₄NO₄: 482.3265, found: 482.3267.

1-((2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-methylnaphthalen-1-yl)methyl)piperidine-4-carboxylic acid (7). Synthesized by general procedure G from 6. White solid; yield: 65%; MP: 151−153 °C; ¹H NMR (400 MHz, Methanol-d₄) δ 8.07 – 8.01 (m, 2H), 7.60 – 7.56 (m, 1H), 7.46 – 7.42 (m, 1H), 7.34 (s, 1H), 4.56 (s, 2H), 4.51 – 4.43 (m, 1H), 3.44 – 3.39 (m, 2H), 3.04 – 2.98 (m, 2H), 2.73 (s, 3H), 2.35 – 2.22 (m, 3H), 1.98 – 1.88 (m, 6H), 1.56 – 1.46 (m, 2H), 1.31 – 1.22 (m, 2H), 1.17 – 1.10 (m, 1H), 0.91 (s, 9H). ¹³C NMR (100 MHz, Methanol-d₄) δ 181.60, 156.25, 140.27, 134.99, 129.65, 128.38, 125.89, 124.91, 124.35, 116.97, 111.40, 78.99, 53.97, 52.25, 48.68, 33.98, 33.15, 28.08, 27.82, 26.58, 20.10. HRMS (ESI): m/z calculated for C₂₈H₄₀NO₃ [M + H]⁺: 438.3003. Found: 438.3000.

1-((2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-fluoronaphthalen-1-yl)methyl)piperidine-4-carboxylic acid (7a). Synthesized by general procedure G from 6a. White solid; yield: 71%; MP: 160−162 °C; ¹H NMR (400 MHz, Chloroform-d) δ 8.24 (d, J = 8.7 Hz, 1H), 7.96 (d, J = 8.3 Hz, 1H), 7.48 (t, J = 7.8 Hz, 1H), 7.33 (t, J = 7.7 Hz, 1H), 6.95 (d, J = 12.1 Hz, 1H), 4.36 (s, 2H), 4.22 – 4.11 (m, 1H), 3.30 – 3.28 (m, 2H), 2.39 (t, J = 11.9 Hz, 2H), 2.16 (d, J = 12.6 Hz, 3H), 1.94 – 1.78 (m, 6H), 1.42 (q, J = 11.7 Hz, 2H), 1.14 – 1.06 (m, 3H), 0.87 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 179.5, 160.0 (d, J_C-F = 250.0 Hz), 155.0 (d, J_C-F = 11.0 Hz), 135.0 (d, J_C-F = 5.0 Hz), 128.0, 124.3, 123.9, 120.6 (d, J_C-F = 2.0 Hz), 119.1 (d, J_C-F = 16.0 Hz), 110.1, 99.6 (d, J_C-F = 23.0 Hz), 78.3, 52.2, 49.7, 47.1, 32.7, 32.3, 27.6, 27.3, 25.4. HRMS (ESI) m/z [M+Na]⁺ calcd for C₂₇H₃₆FNO₃Na: 464.2571, found: 464.2569.

1-((2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-methoxynaphthalen-1-yl)methyl)piperidine-4-carboxylic acid (7b). Synthesized by general procedure G from 6b. Yellow solid; yield: 63%; MP: 137−139°C; ¹H NMR (400 MHz, Chloroform-d) δ 8.18 – 8.13 (m, 2H), 7.44 (t, J = 7.8 Hz, 1H), 7.29 (t, J = 7.8 Hz, 1H), 6.58 (s, 1H), 4.38 (s, 2H), 4.25 – 4.18 (m, 1H), 3.99 (s, 3H), 3.32 (s, 2H), 2.36 (s, 2H), 2.17 (d, J = 12.1 Hz, 2H), 2.06 – 2.03 (m, 1H), 1.95 – 1.79 (m, 6H), 1.49 – 1.46 (m, 2H), 1.16 – 1.09 (m, 3H), 0.87 (s, 9H). ¹³C NMR (100 MHz, Chloroform-d) δ 173.8, 157.3, 155.6, 134.8, 127.7, 124.0, 123.2, 122.0, 121.5, 110.0, 94.8, 55.5, 52.1, 52.0, 47.1, 32.9, 32.3, 27.6, 27.4, 25.5. HRMS (ESI) m/z [M+H]⁺ calcd for C₂₈H₄₀NO₄: 454.2952, found: 454.2951.

2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-methyl-7-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1-naphthaldehyde (9). Synthesized by general procedure H from 8. Yellow oil; yield: 46%; MP: 89−91 °C; ¹H NMR (400 MHz, Chloroform-d) δ 10.85 (s, 1H), 9.74 (s, 1H), 7.90 (d, J = 8.4 Hz, 1H), 7.80 (dd, J = 8.3, 1.2 Hz, 1H), 7.16 (s, 1H), 4.41 – 4.34 (m, 1H), 2.73 (s, 3H), 2.25 – 2.21 (m, 2H), 1.92 – 1.88 (m, 2H), 1.53 – 1.48 (m, 2H), 1.38 (s, 12H), 1.22 – 1.09 (m, 3H), 0.89 (s, 9H).

2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-7-hydroxy-4-methyl-1-naphthaldehyde (10). Synthesized by general procedure I from 9. White solid; yield: 42%; MP: 261−263 °C; ¹H NMR (400 MHz, DMSO-d₆) δ 10.65 (s, 1H), 10.00 (s, 1H), 8.66 (s, 1H), 7.85 (d, J = 9.1 Hz, 1H), 7.23 (s, 1H), 6.99 (d, J = 9.2 Hz, 1H), 4.55 (s, 1H), 2.64 (s, 3H), 2.17 – 2.14 (m, 2H), 1.80 – 1.77 (m, 2H), 1.41 (q, J = 12.2 Hz, 2H), 1.22 – 1.04 (m, 3H), 0.85 (s, 9H). ¹³C NMR (100 MHz, DMSO-d₆) δ 190.60, 163.56, 158.88, 145.85, 126.38, 121.90, 116.21, 114.32, 113.20, 106.97, 77.66, 46.46, 32.16, 32.11, 27.52, 24.92, 20.16.

2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-7-methoxy-4-methyl-1-naphthaldehyde (11a). Synthesized by general procedure J from 10. White solid; yield: 78%; MP: 154−156 °C; ¹H NMR (400 MHz, Chloroform-d) δ 10.83 (s, 1H), 8.94 (d, J = 2.6 Hz, 1H), 7.83 (d, J = 9.1 Hz, 1H), 7.08 (dd, J = 9.2, 2.7 Hz, 1H), 6.96 (s, 1H), 4.42 – 4.34 (m, 1H), 3.97 (s, 3H), 2.69 (s, 3H), 2.26 – 2.12 (m, 2H), 1.92 – 1.88 (m, 2H), 1.56 – 1.47 (m, 2H), 1.19 – 1.09 (m, 3H), 0.89 (s, 9H).

2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-7-(2-fluoroethoxy)-4-methyl-1-naphthaldehyde (11b). Synthesized by general procedure K from 10. White solid; yield: 86%; MP: 151−153 °C; ¹H NMR (400 MHz, Chloroform-d) δ 10.82 (s, 1H), 8.94 (d, J = 2.6 Hz, 1H), 7.84 (d, J = 9.2 Hz, 1H), 7.13 (dd, J = 9.2, 2.7 Hz, 1H), 6.97 (s, 1H), 4.90 – 4.88 (m, 1H), 4.79 – 4.77 (m, 1H), 4.45– 4.43 (m, 1H), 4.42 – 4.34 (m, 2H), 2.69 (s, 3H), 2.26 – 2.22 (m, 2H), 1.93 – 1.88 (m, 2H), 1.56 – 1.47 (m, 2H), 1.22 – 1.09 (m, 3H), 0.89 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 192.17, 163.86, 159.60, 145.24, 133.75, 125.75, 123.21, 116.97, 113.97, 104.66, 81.85 (d, J_C-F = 170.0 Hz), 78.84, 67.01 (d, J_C-F = 21.0 Hz), 47.08, 32.64, 32.33, 27.61, 25.46, 20.85.

1-((2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-7-methoxy-4-methylnaphthalen-1-yl) methyl) piperidine-4-carboxylic acid (12a). Synthesized by general procedure L from 11a. Brown solid; yield: 12%; MP: 130−132 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.68 (d, J = 9.1 Hz, 1H), 7.41 (s, 1H), 6.92 – 6.88 (m, 2H), 4.19 – 4.17(m, 3H), 3.80 (s, 3H), 3.10 (s, 2H), 2.55 (s, 3H), 2.24 – 2.14 (m, 4H), 1.96 (s, 1H), 1.88 – 1.65 (m, 6H), 1.41 (q, J = 11.7 Hz, 2H), 1.11 – 1.05 (m, 3H), 0.85 (s, 9H). HRMS (ESI) m/z [M+H]⁺ calcd for C₂₉H₄₂NO₄: 468.3108, found: 468.3106.

1-((2-(((trans)-4-(tert-butyl)cyclohexyl)oxy)-7-(2-fluoroethoxy)-4-methylnaphthalen-1-yl) methyl)piperidine-4-carboxylic acid (12b). Synthesized by general procedure L from 11b. Brown solid; yield: 24%; MP: 188−190 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.80 – 7.75 (m, 1H), 7.60 – 7.52 (m, 1H), 7.03 – 6.93 (m, 2H), 4.77 – 4.75 (m, 2H), 4.37 – 4.25 (m, 5H), 3.23 (s, 2H), 2.62 (s, 3H), 2.40 (s, 2H), 2.19 – 2.07 (m, 3H), 1.86 – 1.65 (m, 6H), 1.44 – 1.41 (m, 2H), 1.15 – 1.10 (m, 3H), 0.88 (s, 9H). HRMS (ESI) m/z [M+H]⁺ calcd for C₃₀H₄₃FNO₄: 500.3171, found: 500.3176.

3-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-6-fluoro-1-methylnaphthalene (13). Synthesized by general procedure C from 8. White solid; yield: 81%; MP: 81−83 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.86 (dd, J = 9.3, 5.9 Hz, 1H), 7.31 (dd, J = 10.3, 2.6 Hz, 1H), 7.10 (td, J = 8.7, 2.6 Hz, 1H), 6.94 (s, 2H), 4.30 – 4.22 (m, 1H), 2.62 (s, 3H), 2.29 – 2.25 (m, 2H), 1.91 – 1.87 (m, 2H), 1.48 – 1.39 (m, 2H), 1.24 – 1.09 (m, 3H), 0.90 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 161.07 (d, J_C-F = 244.0 Hz), 156.20, 136.36, 136.04 (d, J_C-F = 10.0 Hz), 126.32 (d, J_C-F = 9.0 Hz), 125.10, 123.59 (d, J_C-F = 70.0 Hz), 119.66, 112.98 (d, J_C-F = 24.0 Hz), 110.30 (d, J_C-F = 21.0 Hz), 105.78 (d, J_C-F = 5.0 Hz), 76.34, 47.28, 32.36, 32.27, 27.65, 25.54, 19.44.

2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-7-fluoro-4-methyl-1-naphthaldehyde (14). Synthesized by general procedure E from 13. Brown solid; yield: 58%; MP: 121−123 °C;¹H NMR (400 MHz, Chloroform-d) δ 10.79 (s, 1H), 9.11 (dd, J = 12.7, 2.7 Hz, 1H), 7.90 (dd, J = 9.1, 6.0 Hz, 1H), 7.21 – 7.18 (m, 1H), 7.07 (s, 1H), 4.42 – 4.37 (m, 1H), 2.71 (s, 3H), 2.26 – 2.21 (m, 2H), 1.92 – 1.89 (m, 2H), 1.57 – 1.47 (m, 2H), 1.22 – 1.09 (m, 3H), 0.89 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 191.64, 163.79, 163.52 (d, J_C-F = 246.0 Hz), 158.30 (d, J_C-F = 9.0 Hz), 145.17, 133.40 (d, J_C-F = 12.0 Hz), 126.34 (d, J_C-F = 9.0 Hz), 124.32 (d, J_C-F = 65.0 Hz), 115.34, 114.29 (d, J_C-F = 25.0 Hz), 109.74 (d, J_C-F = 24.0 Hz), 78.88, 47.02, 32.60, 32.33, 27.60, 25.43, 20.99.

Ethyl 1-((2-(((trans)-4-(tert-butyl)cyclohexyl)oxy)-7-fluoro-4-methylnaphthalen-1-yl)methyl) piperidine-4-carboxylate (15). Synthesized by general procedure M from 14. Yellow oil; yield: 53%; ¹H NMR (400 MHz, Chloroform-d) δ 7.89 – 7.84 (m, 2H), 7.12 (td, J = 8.6, 2.5 Hz, 1H), 7.02 (s, 1H), 4.24 – 4.17 (m, 1H), 4.10 (q, J = 7.1 Hz, 2H), 3.81 (s, 2H), 2.89 (d, J = 11.3 Hz, 2H), 2.65 (s, 3H), 2.25 (d, J = 11.9 Hz, 1H), 2.20 – 2.09 (m, 4H), 1.88 – 1.80 (m, 4H), 1.72 – 1.64 (m, 2H), 1.48 – 1.42 (m, 2H), 1.24 – 1.21 (m, 3H), 1.15 – 1.07 (m, 3H), 0.88 (s, 9H).

1-((2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-7-fluoro-4-methylnaphthalen-1-yl) methyl) piperidine-4-carboxylic acid (12c). Synthesized by general procedure G from 15. Yellow solid; yield: 62%; MP: 258−260 °C; ¹H NMR (400 MHz, Chloroform-d) δ 8.05 (d, J = 11.8 Hz, 1H), 7.86 (dd, J = 9.2, 5.9 Hz, 1H), 7.12 – 7.03 (m, 2H), 4.41 (s, 2H), 4.29 (s, 1H), 3.35 (s, 2H), 2.66 (s, 3H), 2.49 – 2.43 (m, 2H), 2.19 (d, J = 12.2 Hz, 3H), 2.01 – 1.98 (m, 2H), 1.89 – 1.82 (s, 4H), 1.49 – 1.40 (m, 2H), 1.17 – 1.12 (m, 3H), 0.88 (s, 9H). HRMS (ESI) m/z [M+H]⁺ calcd for C₂₈H₃₉FNO₃: 456.2908, found: 456.2904.

3-(((2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-methylnaphthalen-1-yl)methyl)amino) propanoic acid (16a). Synthesized by general procedure N from 5 and methyl 3-aminopropanoate hydrochloride. White solid; yield: 60%; MP: 103−105 °C; ¹H NMR (400 MHz, Chloroform-d) δ 8.00 (d, J = 8.4 Hz, 1H), 7.87 (d, J = 8.3 Hz, 1H), 7.40 (t, J = 7.5 Hz, 1H), 7.30 (t, J = 7.5 Hz, 1H), 7.08 (s, 1H), 4.51 (s, 2H), 4.32 – 4.24 (m, 1H), 2.90 (t, J = 5.9 Hz, 2H), 2.68 (s, 3H), 2.40 (t, J = 5.9 Hz, 2H), 2.21 – 2.17 (m, 2H), 1.81 (d, J = 11.6 Hz, 2H), 1.48 (q, J = 11.8 Hz, 2H), 1.15 – 1.03 (m, 3H), 0.84 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 176.93, 154.25, 137.87, 133.35, 127.99, 127.27, 124.57, 123.52, 122.97, 115.31, 111.42, 77.47, 46.97, 43.09, 40.17, 32.58, 32.25, 31.79, 27.56, 25.43, 20.13. HRMS (ESI) m/z [M+H]⁺ calcd for C₂₅H₃₆NO₃: 398.2690, found: 3987.2689.

3-(((2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-methylnaphthalen-1-yl)methyl)amino)-2-methylpropanoic acid (16b). Synthesized by general procedure N from 5 and ethyl 3-amino-2-methylpropanoate hydrochloride. White solid; yield: 44%; MP: 130−132 °C; ¹H NMR (400 MHz, Chloroform-d) δ 8.01 – 7.86 (m, 2H), 7.43 – 7.42 (m, 2H), 7.08 (s, 1H), 4.59 (s, 2H), 4.29 (s, 1H), 2.95 – 2.66 (m, 5H), 2.20 – 2.13 (m, 2H), 1.84 (d, J = 17.3 Hz, 3H), 1.44 (t, J = 13.0 Hz, 2H), 1.15 – 1.01 (m, 6H), 0.85 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 179.68, 154.45, 138.27, 133.23, 128.11, 127.33, 124.53, 123.70, 123.19, 115.45, 110.94, 77.79, 48.72, 46.96, 40.14, 36.68, 32.56, 32.25, 27.56, 25.42, 20.13, 15.65. HRMS (ESI) m/z [M+H]⁺ calcd for C₂₆H₃₈NO₃: 412.2846, found: 412.2841.

3-(((2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-methylnaphthalen-1-yl)methyl)amino)-2-fluoropropanoic acid (16c). Synthesized by general procedure O from 5 and 3-amino-2-fluoropropanoic acid hydrochloride salts. Yellow solid; yield: 53%; MP: 180−182 °C; ¹H NMR (400 MHz, CD₃OD) δ 8.06 (d, J = 8.6 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.58 (t, J = 7.6 Hz, 1H), 7.47 – 7.41 (m, 1H), 7.33 (s, 1H), 5.09 – 4.93 (m, 1H), 4.63 (s, 2H), 4.52 – 4.44 (m, 1H), 3.53 – 3.34 (m, 2H), 2.72 (s, 3H), 2.29 – 2.23 (m, 2H), 1.90 (d, J = 12.9 Hz, 2H), 1.58 – 1.48 (m, 2H), 1.31 – 1.12 (m, 2H), 1.17 – 1.09 (m, 1H), 0.91 (s, 9H); ¹³C NMR (100 MHz, CD₃OD) δ 172.60, 154.33, 138.47, 132.95, 128.17, 127.06, 124.56, 123.47, 122.22, 115.44, 111.68, 86.58 (d, J_C-F = 183.0 Hz), 77.59, 49.16 (d, J_C-F = 23.0 Hz), 47.20, 41.79, 32.47, 31.72, 26.66, 25.15, 18.68. HRMS (ESI) m/z [M+H]⁺ calcd for C₂₅H₃₄FNO₃: 438.2415, found: 438.2412.

4-(((2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-methylnaphthalen-1-yl)methyl)(methyl)amino) butanoic acid (16d). Synthesized by general procedure P from 5 and ethyl 4-aminobutanoate hydrochloride. White solid; yield: 25%; MP: 85−87 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.97 (d, J = 8.5 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.54 (t, J = 7.7 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.11 (s, 1H), 4.33 – 4.27 (m, 3H), 2.84 (t, J = 5.5 Hz, 2H), 2.69 (s, 3H), 2.47 (s, 3H), 2.38 – 2.35 (m, 2H), 2.23 – 2.19 (m, 2H), 1.89 – 1.81 (m, 4H), 1.44 (q, J = 11.3, 10.7 Hz, 2H), 1.21 – 1.08 (m, 3H), 0.88 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 173.77, 154.4, 137.81, 133.75, 128.08, 127.19, 124.62, 123.67, 123.44, 115.77, 112.81, 77.68, 57.87, 50.32, 47.19, 40.89, 37.11, 32.88, 32.32, 27.60, 25.49, 21.26, 20.12. HRMS (ESI) m/z [M+H]⁺ calcd for C₂₇H₄₀NO₃: 426.3003, found: 426.3001.

3-((2-((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-methylnaphthalen-1-yl)methyl)(methyl)amino) propanoic acid (16e). Synthesized by general procedure P from 5 and methyl 3-aminopropanoate hydrochloride. White solid; yield: 34%; MP: 84−86 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.95 – 7.93 (m, 2H), 7.54 (t, J = 7.5 Hz, 1H), 7.39 (t, J = 7.5 Hz, 1H), 7.12 (s, 1H), 4.35 – 4.29 (m, 1H), 4.23 (s, 2H), 2.97 – 2.94 (m, 2H), 2.70 (s, 3H), 2.52 – 2.49 (m, 2H), 2.43 (s, 3H), 2.24 – 2.21 (m, 2H), 1.89 – 1.86 (m, 2H), 1.48 (q, J = 11.9 Hz, 2H), 1.18 – 1.09 (qm, 3H), 0.88 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 174.2, 154.3, 137.6, 133.7, 128.0, 127.2, 124.8, 123.5, 122.8, 115.6, 113.1, 77.5, 52.6, 50.4, 47.1, 39.6, 32.8, 32.3, 29.8, 27.6, 25.5, 20.1. HRMS (ESI) m/z [M+H]⁺ calcd for C₂₆H₃₈NO₃: 412.2846, found: 412.2841.

N-((2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-methylnaphthalen-1-yl)methyl)-N-methyl glycine (16f). Synthesized by general procedure P from 5 and methyl glycinate hydrochloride. White solid; yield: 51%; MP: 100−102 °C; ¹H NMR (400 MHz, Chloroform-d) δ 8.26 (d, J = 8.5 Hz, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.36 – 7.30 (m, 1H), 7.10 (s, 1H), 4.73 (s, 2H), 4.37 – 4.29 (m, 1H), 3.62 (s, 2H), 2.74 (s, 3H), 2.68 (s, 3H), 2.22 – 2.17 (m, 2H), 1.87 – 1.84 (m, 2H), 1.53 – 1.43 (m, 2H), 1.19 – 1.03 (m, 3H), 0.86 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 168.39, 154.80, 139.02, 133.73, 128.05, 127.69, 124.64, 123.84, 123.26, 115.25, 110.00, 77.85, 59.33, 50.40, 46.94, 41.58, 32.66, 32.25, 27.54, 25.42, 20.16. HRMS (ESI) m/z [M+Na]⁺ calcd for C₂₅H₃₅NO₃Na: 420.2509, found: 420.2503.

3-(((2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-methylnaphthalen-1-yl)methyl)amino)propan-1-ol (16g). Synthesized by general procedure Q from 5 and 3-aminopropan-1-ol. Yellow oil; yield: 43%; ¹H NMR (400 MHz, Chloroform-d) δ 8.02 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.39 (t, J = 7.5 Hz, 1H), 7.11 (s, 1H), 4.28 – 4.20 (m, 3H), 3.82 (t, J = 5.3 Hz, 2H), 2.92 (t, J = 5.5 Hz, 2H), 2.68 (s, 3H), 2.23 – 2.19 (m, 2H), 1.90 – 1.86 (m, 2H), 1.70 (p, J = 5.3 Hz, 2H), 1.46 (q, J = 11.9 Hz, 2H), 1.20 – 1.04 (m, 3H), 0.89 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 153.08, 135.44, 133.52, 128.42, 126.46, 124.62, 123.42, 123.33, 120.06, 117.13, 78.18, 64.71, 49.65, 47.24, 43.10, 32.99, 32.33, 30.59, 27.63, 25.54, 19.88. HRMS (ESI) m/z [M+H]⁺ calcd for C₂₅H₃₈NO₂: 384.2897, found: 384.2895.

3-(((2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-methylnaphthalen-1-yl)methyl)amino)-2-fluoropropan-1-ol (16h). Synthesized by general procedure Q from 5 and 3-amino-2-fluoropropan-1-ol. Yellow oil; yield: 70%; ¹H NMR (400 MHz, Chloroform-d) δ 8.01 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.39 (t, J = 7.5 Hz, 1H), 7.12 (s, 1H), 4.74 – 4.60 (m, 1H), 4.32 – 4.23 (m, 3H), 3.92 – 3.77 (m, 2H), 3.15 – 2.96 (m, 2H), 2.69 (s, 3H), 2.23 – 2.19 (m, 2H), 1.90 – 1.86 (m, 2H), 1.46 (q, J = 12.9, 12.0 Hz, 2H), 1.20 – 1.09 (m, 3H), 0.89 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 153.27, 135.97, 133.45, 128.40, 126.67, 124.70, 123.43, 123.25, 118.65, 116.80, 91.45 (d, J_C-F = 170.0 Hz), 78.11, 64.76 (d, J_C-F = 23.0 Hz), 50.51 (d, J_C-F = 22.0 Hz), 47.20, 43.36, 32.94, 32.35, 27.65, 25.55, 19.97. HRMS (ESI) m/z [M+H]⁺ calcd for C₂₅H₃₇FNO₂: 402.2803, found: 402.2798.

1-(((2-((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-methylnaphthalen-1-yl)methyl)amino)-3-fluoropropan-2-ol (16i). Synthesized by general procedure Q from 5 and 1-amino-3-fluoropropan-2-ol. Yellow oil; yield: 67%; ¹H NMR (400 MHz, Chloroform-d) δ 7.99 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 8.3 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.40 (t, J = 7.5 Hz, 1H), 7.12 (s, 1H), 4.44 – 4.24 (m, 5H), 2.93 – 2.89 (m, 1H), 2.77 – 2.72 (m, 1H), 2.68 (s, 3H), 2.24 – 2.18 (m, 2H), 1.88 (d, J = 12.7 Hz, 2H), 1.52 – 1.43 (m, 2H), 1.20 – 1.09 (m, 3H), 0.88 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 153.46, 136.59, 133.25, 128.32, 126.89, 124.75, 123.53, 122.99, 116.79, 116.46, 84.97 (d, J_C-F = 170.0 Hz), 78.15, 67.16 (d, J_C-F = 20.0 Hz), 49.46 (d, J_C-F = 7.0 Hz), 47.09, 42.70, 32.85, 32.31, 27.61, 25.52, 19.99. HRMS (ESI) m/z [M+H]⁺ calcd for C₂₅H₃₇FNO₂: 402.2803, found: 402.2799.

3-(((2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-methylnaphthalen-1-yl)methyl)amino) propane-1-sulfonic acid (16j). Synthesized by general procedure R from 5 and 3-aminopropane-1-sulfonic acid. White solid; yield: 55%; MP: 180−182 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.82 (d, J = 8.4 Hz, 1H), 7.69 (d, J = 8.3 Hz, 1H), 7.35 (t, J = 7.4 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 7.02 (s, 1H), 4.38 (s, 2H), 4.33 – 4.26 (m, 1H), 2.99 (t, J = 7.0 Hz, 2H), 2.73 (t, J = 6.8 Hz, 2H), 2.50 (s, 3H), 2.20 – 2.16 (m, 2H), 2.09 – 2.05 (m, 2H), 1.80 (d, J = 11.2 Hz, 2H), 1.47 (q, J = 10.8, 9.9 Hz, 2H), 1.16 – 1.03 (m, 3H), 0.83 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 154.59, 138.59, 132.95, 127.83, 127.58, 124.43, 123.75, 123.03, 115.31, 109.83, 77.85, 48.45, 46.83, 46.31, 41.45, 32.57, 32.25, 27.57, 25.44, 21.45, 20.02. HRMS (ESI) m/z [M+Na]⁺ calcd for C₂₅H₃₇SNO₄Na: 470.2336, found: 470.2333.

3-(((2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-methylnaphthalen-1-yl)methyl)amino)-N-methyl propane-1-sulfonamide (16k). Synthesized by general procedure S from 5 and 3-amino-N-methylpropane-1-sulfonamide hydrochloride. Yellow solid; yield: 40%; MP: 125−127 °C; ¹H NMR (400 MHz, Chloroform-d) δ 8.02 (d, J = 8.5 Hz, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.12 (s, 1H), 4.50 (s, 2H), 4.37 – 4.30 (m, 1H), 3.06 (t, J = 6.9 Hz, 2H), 2.97 (t, J = 6.6 Hz, 2H), 2.68 (s, 3H), 2.65 (s, 3H), 2.29 – 2.20 (m, 4H), 1.90 – 1.86 (m, 2H), 1.51 (q, J = 11.6 Hz, 2H), 1.21 – 1.10 (m, 3H), 0.88 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 174.29, 154.38, 138.32, 133.05, 128.11, 127.54, 124.78, 123.85, 122.90, 115.68, 78.19, 48.09, 46.96, 45.59, 41.83, 32.72, 32.31, 28.99, 27.60, 25.49, 21.38, 20.15. HRMS (ESI) m/z [M+H]⁺ calcd for C₂₆H₄₁SN₂O₃: 461.2832, found: 461.2830.

N-((2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-methylnaphthalen-1-yl)methyl)-3-fluoro propan-1-amine (16l). Synthesized by general procedure S from 5 and 3-fluoropropan-1-amine hydrochloride. Yellow oil; yield: 86%; ¹H NMR (400 MHz, Chloroform-d) δ 8.04 (d, J = 8.5 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.12 (s, 1H), 4.52 (t, J = 5.7 Hz, 1H), 4.42 – 4.38 (m, 3H), 4.34 – 4.26 (m, 1H), 2.92 (t, J = 7.1 Hz, 2H), 2.67 (s, 3H), 2.25 – 2.20 (m, 2H), 2.07 – 1.94 (m, 2H), 1.91 – 1.86 (m, 2H), 1.53 – 1.43 (m, 2H), 1.21 – 1.09 (m, 3H), 0.88 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 153.74, 136.93, 133.33, 128.30, 127.03, 124.68, 123.61, 123.19, 116.32, 109.99, 82.30 (d, J_C-F = 163.0 Hz), 47.14, 44.2 (d, J_C-F = 5.0 Hz), 42.57, 32.86, 32.35, 29.08 (d, J_C-F = 19.0 Hz), 27.64, 25.55, 20.06. HRMS (ESI) m/z [M+H]⁺ calcd for C₂₅H₃₇FNO: 386.2854, found: 386.2850.

N-((2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-methylnaphthalen-1-yl)methyl)-3-(methylthio) propan-1-amine (16m). Synthesized by general procedure Q from 5 and 3-(methylthio)propan-1-amine. Yellow oil; yield: 85%; ¹H NMR (400 MHz, Chloroform-d) δ 8.05 (d, J = 8.5 Hz, 1H), 7.91 (d, J = 8.4 Hz, 1H), 7.53 (t, J = 7.6 Hz, 1H), 7.40 (s, 1H), 7.12 (s, 1H), 4.38 (s, 2H), 4.34 – 4.26 (m, 1H), 2.87 (t, J = 7.1 Hz, 2H), 2.67 (s, 3H), 2.48 (t, J = 7.0 Hz, 2H), 2.25 – 2.20 (m, 2H), 2.04 (s, 3H), 1.96 – 1.85 (m, 4H), 1.54 – 1.45 (m, 2H), 1.21 – 1.06 (m, 3H), 0.88 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 153.74, 136.92, 133.30, 128.25, 127.01, 124.62, 123.58, 123.23, 116.29, 78.16, 47.10, 46.96, 42.33, 32.87, 32.30, 31.66, 27.60, 26.91, 25.51, 20.02, 15.21. HRMS (ESI) m/z [M+H]⁺ calcd for C₂₆H₄₀SNO: 414.2825, found: 414.2823.

N-((2-((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-methylnaphthalen-1-yl)methyl)-3-(2-fluoro ethoxy)propan-1-amine (16n). Synthesized by general procedure S from 5 and 3-(2-fluoroethoxy)propan-1-amine hydrochloride. Yellow oil; yield: 90%; ¹H NMR (400 MHz, Chloroform-d) δ 8.03 (d, J = 8.5 Hz, 1H), 7.92 (d, J = 8.4 Hz, 1H), 7.55 (t, J = 7.6 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.12 (s, 1H), 4.50 (s, 2H), 4.41 – 4.39 (m, 1H), 4.36 – 4.27 (m, 2H), 3.56 – 3.54 (m, 1H), 3.51 – 3.47 (m, 3H), 2.99 (t, J = 6.4 Hz, 2H), 2.68 (s, 3H), 2.22 – 2.18 (m, 2H), 1.97 (p, J = 6.1 Hz, 2H), 1.90 – 1.85 (m, 2H), 1.48 (q, J = 12.1 Hz, 2H), 1.20 – 1.05 (m, 3H), 0.87 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 154.14, 137.92, 133.09, 128.19, 127.45, 124.76, 123.79, 122.78, 115.95, 112.58, 82.75 (d, J_C-F = 168.0 Hz), 78.27, 69.89 (d, J_C-F = 19.0 Hz), 69.74, 46.99, 45.70, 41.95, 32.75, 32.26, 27.55, 26.63, 25.45, 20.07. HRMS (ESI) m/z [M+H]⁺ calcd for C₂₇H₄₁FNO₂: 430.3116, found: 430.3112.

1-((2-((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-methylnaphthalen-1-yl)methyl)-4-methyl piperazine (16o). Synthesized by general procedure T from 5 and tert-butyl piperazine-1-carboxylate. White solid; yield: 21%; MP: 108−110 °C; ¹H NMR (400 MHz, Chloroform-d) δ 8.24 (d, J = 8.5 Hz, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.47 (t, J = 8.0 Hz, 1H), 7.37 (t, J = 7.9 Hz, 1H), 7.08 (s, 1H), 4.23 – 4.15 (m, 1H), 3.92 (s, 2H), 2.67 – 2.25 (m, 14H), 2.21 – 2.16 (m, 2H), 1.86 – 1.83 (m, 2H), 1.44 (q, J = 11.1 Hz, 2H), 1.17 – 1.06 (m, 3H), 0.88 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 153.52, 135.26, 134.61, 128.48, 125.78, 125.49, 124.06, 123.25, 118.84, 117.39, 78.26, 55.32, 52.97, 51.93, 47.32, 46.05, 33.00, 32.34, 27.65, 25.60, 19.81. HRMS (ESI) m/z [M+H]⁺ calcd for C₂₇H₄₁N₂O: 409.3213, found: 409.3209.

1-((2-(((trans)-4-(tert-Butyl)cyclohexyl)oxy)-4-methylnaphthalen-1-yl)methyl)-4-(2-fluoro ethyl)piperazine (16p). Synthesized by general procedure T from 5 and tert-butyl piperazine-1-carboxylate. Colorless oil; yield: 27%; ¹H NMR (400 MHz, Chloroform-d) δ 8.23 (d, J = 8.5 Hz, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.47 (t, J = 7.6 Hz, 1H), 7.37 (t, J = 7.5 Hz, 1H), 7.09 (s, 1H), 4.61 (t, J = 5.0 Hz, 1H), 4.49 (t, J = 5.0 Hz, 1H), 4.24 – 4.16 (m, 1H), 3.94 (s, 2H), 2.71 – 2.50 (m, 13H), 2.21 – 2.16 (m, 2H), 1.87 – 1.84 (m, 2H), 1.48 – 1.40 (m, 2H), 1.17 – 1.03 (m, 3H), 0.88 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 153.55, 135.38, 134.58, 125.82, 125.40, 124.08, 123.25, 117.24, 81.89 (d, J_C-F = 167.0 Hz), 78.10, 58.20 (d, J_C-F = 167.0 Hz), 53.64, 52.74, 51.80, 47.30, 32.99, 32.33, 27.64, 25.58, 19.82. HRMS (ESI) m/z [M+H]⁺ calcd for C₂₈H₄₂FN₂O: 441.3276, found: 441.3272.

2-Methoxy-4-methyl-1-naphthaldehyde (17a). Synthesized by general procedure E from 3-methoxy-1-methylnaphthalene. White solid; yield: 80%; MP: 135−137 °C; ¹H NMR (400 MHz, Chloroform-d) δ 10.85 (s, 1H), 9.36 (d, J = 8.7 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.62 (t, J = 8.3 Hz, 1H), 7.44 (t, J = 8.2 Hz, 1H), 7.13 (s, 1H), 4.03 (s, 3H), 2.75 (s, 3H); ¹³C NMR (100 MHz, Chloroform-d) δ 191.52, 163.65, 145.63, 131.78, 129.50, 127.70, 125.48, 124.55, 124.05, 115.18, 113.55, 56.42, 20.91.

2-(2-Fluoroethoxy)-4-methyl-1-naphthaldehyde (17b). Synthesized by general procedure E from 3-(2-fluoroethoxy)-1-methylnaphthalene. White solid; yield: 79%; MP: 122−124 °C; ¹H NMR (400 MHz, Chloroform-d) δ 10.90 (s, 1H), 9.36 (d, J = 9.0 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.63 (t, J = 8.3 Hz, 1H), 7.47 (t, J = 8.1 Hz, 1H), 7.10 (s, 1H), 4.90 – 4.88 (m, 1H), 4.78 – 4.76 (m, 1H), 4.50 – 4.48 (m, 1H), 4.43 – 4.41 (m, 1H), 2.75 (s, 3H); ¹³C NMR (100 MHz, Chloroform-d) δ 191.47, 162.52, 145.61, 131.66, 129.58, 128.14, 125.64, 124.93, 124.07, 116.08, 114.71, 81.61 (d, J_C-F = 172.0 Hz), 68.82 (d, J_C-F = 21.0 Hz), 20.85.

2-(3-Fluoropropoxy)-4-methyl-1-naphthaldehyde (17c). Synthesized by general procedure E from 3-(3-fluoropropoxy)-1-methylnaphthalene. White solid; yield: 81%; MP: 102−104 °C; ¹H NMR (400 MHz, Chloroform-d) δ 10.79 (s, 1H), 9.32 (d, J = 8.7 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.58 (t, J = 7.7 Hz, 1H), 7.41 (t, J = 7.6 Hz, 1H), 7.00 (s, 1H), 4.72 (t, J = 5.7 Hz, 1H), 4.61 (t, J = 5.7 Hz, 1H), 4.25 (t, J = 6.1 Hz, 2H), 2.64 (s, 3H), 2.25 (p, J = 5.9 Hz, 1H), 2.18 (p, J = 5.9 Hz, 1H); ¹³C NMR (100 MHz, Chloroform-d) δ 191.01, 162.74, 145.54, 131.49, 129.28, 127.58, 125.26, 124.46, 123.94, 115.04, 114.11, 80.25 (d, J_C-F = 164.0 Hz), 64.82 (d, J_C-F = 5.0 Hz), 30.28 (d, J_C-F = 19.0 Hz), 20.63.

2-(4-Fluorobutoxy)-4-methyl-1-naphthaldehyde (17d). Synthesized by general procedure E from 3-(4-fluorobutoxy)-1-methylnaphthalene. Yellow solid; yield: 83%; MP: 118−120 °C; ¹H NMR (400 MHz, Chloroform-d) δ 10.87 (s, 1H), 9.36 (d, J = 8.7 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.62 (t, J = 7.7 Hz, 1H), 7.45 (t, J = 7.6 Hz, 1H), 7.12 (s, 1H), 4.61 (t, J = 5.5 Hz, 1H), 4.49 (t, J = 5.8 Hz, 1H), 4.26 (t, J = 6.0 Hz, 2H), 2.74 (s, 3H), 2.07 – 1.87 (m, 4H); ¹³C NMR (100 MHz, Chloroform-d) δ 191.38, 163.15, 145.63, 131.75, 129.51, 127.72, 125.48, 124.62, 124.05, 115.34, 114.41, 83.58 (d, J_C-F = 165.0 Hz), 68.75, 27.13 (d, J_C-F = 20.0 Hz), 25.54 (d, J_C-F = 5.0 Hz), 20.88.

2-((5-Fluoropentyl)oxy)-4-methyl-1-naphthaldehyde (17e). Synthesized by general procedure E from 3-((5-fluoropentyl)oxy)-1-methylnaphthalene. Yellow solid; yield: 84%; MP: 98−100 °C; ¹H NMR (400 MHz, Chloroform-d) δ 10.80 (s, 1H), 9.34 (d, J = 8.7 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.57 (t, J = 7.7 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 6.96 (s, 1H), 4.52 (t, J = 6.0 Hz, 1H), 4.40 (t, J = 5.9 Hz, 1H), 4.08 (t, J = 6.3 Hz, 2H), 2.63 (s, 3H), 1.89 – 1.69 (m, 4H), 1.63 – 1.56 (m, 2H); ¹³C NMR (100 MHz, Chloroform-d) δ 191.19, 163.08, 145.42, 131.50, 129.17, 127.38, 125.18, 124.27, 123.88, 114.85, 114.16, 83.64 (d, J_C-F = 164.0 Hz), 68.80, 29.90 (d, J_C-F = 20.0 Hz), 28.74, 21.79 (d, J_C-F = 5.0 Hz), 20.58.

2-((6-Fluorohexyl)oxy)-4-methyl-1-naphthaldehyde (17f). Synthesized by general procedure E from 3-((6-fluorohexyl)oxy)-1-methylnaphthalene. Yellow solid; yield: 84%; MP: 78−80 °C; ¹H NMR (400 MHz, Chloroform-d) δ 10.82 (s, 1H), 9.35 (d, J = 8.6 Hz, 1H), 7.86 (d, J = 8.4 Hz, 1H), 7.58 (t, J = 7.7 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 7.00 (s, 1H), 4.51 (t, J = 6.0 Hz, 1H), 4.39 (t, J = 6.0 Hz, 1H), 4.10 (t, J = 6.3 Hz, 2H), 2.65 (s, 3H), 1.87 – 1.79 (m, 2H), 1.77 – 1.66 (m, 2H), 1.55 – 1.46 (m, 4H); ¹³C NMR (100 MHz, Chloroform-d) δ 191.3, 163.2, 145.4, 131.6, 129.2, 127.4, 125.2, 124.3, 123.9, 114.9, 114.3, 83.8 (d, J_C-F = 164.0 Hz), 68.9, 30.2 (d, J_C-F = 19.0 Hz), 29.0, 25.6, 24.9 (d, J_C-F = 5.0 Hz), 20.6.

Ethyl 3-(((2-methoxy-4-methylnaphthalen-1-yl)methyl)amino)propanoate (18a). Synthesized by general procedure S from 17a and ethyl 3-aminopropanoate hydrochloride. Yellow oil; yield: 60%; ¹H NMR (400 MHz, Chloroform-d) δ 8.03 (d, J = 8.6 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.52 (t, J = 7.3 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.14 (s, 1H), 4.31 (s, 2H), 4.10 (q, J = 7.1 Hz, 2H), 3.97 (s, 3H), 3.00 (t, J = 6.6 Hz, 2H), 2.70 (s, 3H), 2.62 (t, J = 6.5 Hz, 2H), 1.21 (t, J = 7.1 Hz, 3H).

Ethyl 3-(((2-(2-fluoroethoxy)-4-methylnaphthalen-1-yl)methyl)amino)propanoate (18b). Synthesized by general procedure S from 17b and ethyl 3-aminopropanoate hydrochloride. Yellow oil; yield: 55%; ¹H NMR (400 MHz, Chloroform-d) δ 8.07 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.53 (t, J = 7.7 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 7.09 (s, 1H), 4.87 – 4.85 (m, 1H), 4.75 – 4.73 (m, 1H), 4.42 – 4.40 (m, 1H), 4.35 – 4.33 (m, 3H), 4.09 (q, J = 7.1 Hz, 2H), 3.01 (t, J = 6.6 Hz, 2H), 2.68 (s, 3H), 2.60 (t, J = 6.6 Hz, 2H), 1.21 (t, J = 7.1 Hz, 3H); ¹³C NMR (100 MHz, Chloroform-d) δ 172.30, 153.82, 137.17, 133.10, 128.59, 127.10, 124.67, 123.82, 123.22, 115.83, 114.91, 82.00 (d, J_C-F = 170.0 Hz), 68.58 (d, J_C-F = 20.0 Hz), 60.67, 43.70, 42.29, 32.77, 19.95, 14.03.

Ethyl 3-(((2-(2-fluoroethoxy)-4-methylnaphthalen-1-yl)methyl)amino)propanoate (18c). Synthesized by general procedure S from 17c and ethyl 3-aminopropanoate hydrochloride. Yellow oil; yield: 48%; ¹H NMR (400 MHz, Chloroform-d) δ 8.07 (d, J = 8.5 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.39 (t, J = 7.6 Hz, 1H), 7.13 (s, 1H), 4.78 (t, J = 5.7 Hz, 1H), 4.67 (t, J = 5.8 Hz, 1H), 4.29 – 4.22 (m, 4H), 4.10 (q, J = 7.1 Hz, 2H), 2.98 (t, J = 6.6 Hz, 2H), 2.69 (s, 3H), 2.55 (t, J = 6.6 Hz, 2H), 2.29 (q, J = 5.9 Hz, 1H), 2.22 – 2.19 (m, 1H), 1.22 (t, J = 7.1 Hz, 3H); ¹³C NMR (100 MHz, Chloroform-d) δ 172.61, 153.56, 135.69, 133.29, 126.46, 124.53, 123.46, 123.26, 118.91, 115.13, 80.77 (d, J_C-F = 161.0 Hz), 64.88 (d, J_C-F = 5.0 Hz), 60.23, 44.69, 42.85, 34.66, 30.66 (d, J_C-F = 20.0 Hz), 19.79, 14.05.

Ethyl 3-(((2-(4-fluorobutoxy)-4-methylnaphthalen-1-yl)methyl)amino)propanoate (18d). Synthesized by general procedure S from 17d and ethyl 3-aminopropanoate hydrochloride. Yellow oil; yield: 50%; ¹H NMR (400 MHz, Chloroform-d) δ 8.05 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.11 (s, 1H), 4.64 – 4.61 (m, 1H), 4.50 (t, J = 5.7 Hz, 1H), 4.27 (s, 2H), 4.19 – 4.16 (m, 2H), 4.09 (qd, J = 7.2, 1.6 Hz, 2H), 2.97 (t, J = 6.5 Hz, 2H), 2.69 (s, 3H), 2.55 (t, J = 6.6 Hz, 2H), 2.01 – 1.90 (m, 4H), 1.21 (t, J = 7.1 Hz, 3H); ¹³C NMR (100 MHz, Chloroform-d) δ 172.68, 153.79, 135.74, 133.36, 128.32, 126.53, 124.59, 123.45, 123.23, 118.57, 115.10, 83.75 (d, J_C-F = 164.0 Hz), 68.51, 60.31, 44.65, 42.88, 34.62, 27.24 (d, J_C-F = 20.0 Hz), 25.64 (d, J_C-F = 4.0 Hz), 19.89, 14.11.

Ethyl 3-(((2-((5-fluoropentyl)oxy)-4-methylnaphthalen-1-yl)methyl)amino)propanoate (18e). Synthesized by general procedure S from 17e and ethyl 3-aminopropanoate hydrochloride. Yellow oil; yield: 51%; ¹H NMR (400 MHz, Chloroform-d) δ 8.05 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.38 (t, J = 7.5 Hz, 1H), 7.11 (s, 1H), 4.56 (t, J = 6.0 Hz, 1H), 4.44 (t, J = 6.0 Hz, 1H), 4.27 (s, 2H), 4.15 – 4.07 (m, 4H), 2.98 (t, J = 6.6 Hz, 2H), 2.69 (s, 3H), 2.56 (t, J = 6.6 Hz, 2H), 1.95 – 1.75 (m, 4H), 1.70 – 1.62 (m, 2H), 1.21 (t, J = 7.1 Hz, 3H); ¹³C NMR (100 MHz, Chloroform-d) δ 172.69, 153.94, 135.77, 128.29, 126.53, 124.60, 123.41, 123.20, 118.39, 115.13, 83.90 (d, J_C-F = 164.0 Hz), 68.87, 60.34, 44.60, 42.87, 34.55, 30.10 (d, J_C-F = 19.0 Hz), 29.21, 21.97 (d, J_C-F = 5.0 Hz), 19.90, 14.11.

Ethyl 3-(((2-((6-fluorohexyl)oxy)-4-methylnaphthalen-1-yl)methyl)amino)propanoate (18f). Synthesized by general procedure S from 17f and ethyl 3-aminopropanoate hydrochloride. Yellow oil; yield: 53%; ¹H NMR (400 MHz, Chloroform-d) δ 8.06 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 8.4 Hz, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.11 (s, 1H), 4.53 (t, J = 6.1 Hz, 1H), 4.42 (t, J = 6.1 Hz, 1H), 4.28 (s, 2H), 4.14 – 4.07 (m, 4H), 2.97 (t, J = 6.6 Hz, 2H), 2.69 (s, 3H), 2.56 (t, J = 6.6 Hz, 2H), 1.91 – 1.84 (m, 2H), 1.82 – 1.69 (m, 2H), 1.62 – 1.50 (m, 4H), 1.21 (t, J = 7.1 Hz, 3H); ¹³C NMR (100 MHz, Chloroform-d) δ 172.7, 154.0, 135.7, 133.4, 128.3, 126.5, 124.6, 123.4, 123.2, 118.5, 115.2, 84.0 (d, J_C-F = 164.0 Hz), 69.0, 60.3, 44.6, 42.9, 34.6, 30.3 (d, J_C-F = 20.0 Hz), 29.5, 25.8, 25.0 (d, J_C-F = 5.0 Hz), 19.9, 14.1.

3-(((2-Methoxy-4-methylnaphthalen-1-yl)methyl)amino)propanoic acid (19a). Synthesized by general procedure G from 18a. White solid; yield: 70%; MP: 180−182 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.90 (d, J = 8.4 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.35 (t, J = 7.5 Hz, 1H), 7.23 (t, J = 7.5 Hz, 1H), 6.98 (s, 1H), 4.24 (s, 2H), 3.79 (s, 3H), 2.76 (t, J = 5.9 Hz, 2H), 2.56 (s, 3H), 2.27 (d, J = 6.0 Hz, 2H); ¹³C NMR (100 MHz, Chloroform-d) δ 178.87, 173.95, 155.65, 138.28, 133.04, 127.37, 124.71, 123.50, 122.64, 113.28, 109.98, 56.06, 20.10. HRMS (ESI) m/z [M+H]⁺ calcd for C₁₆H₂₀NO₃: 274.1438, found: 274.1435.

3-(((2-(2-Fluoroethoxy)-4-methylnaphthalen-1-yl)methyl)amino)propanoic acid (19b). Synthesized by general procedure G from 18b. Yellow solid; yield: 67%; MP: 120−122 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.87 (d, J = 8.3 Hz, 1H), 7.76 (d, J = 8.3 Hz, 1H), 7.36 (t, J = 7.4 Hz, 1H), 7.23 (t, J = 7.4 Hz, 1H), 6.95 (s, 1H), 4.74 – 4.72 (m, 1H), 4.62 – 4.60 (m, 1H), 4.25 – 4.16 (m, 4H), 2.76 (t, J = 6.0 Hz, 2H), 2.53 (s, 3H), 2.22 (t, J = 5.9 Hz, 2H). HRMS (ESI) m/z [M+H]⁺ calcd for C₁₇H₂₀FNO₃: 328.1319, found: 328.1315.

3-(((2-(3-Fluoropropoxy)-4-methylnaphthalen-1-yl)methyl)amino)propanoic acid (19c). Synthesized by general procedure G from 18c. Yellow solid; yield: 52%; MP: 105−107 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.95 (d, J = 8.5 Hz, 1H), 7.88 (d, J = 8.3 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 7.30 (t, J = 7.5 Hz, 1H), 7.08 (s, 1H), 4.65 (t, J = 5.5 Hz, 1H), 4.54 (t, J = 5.5 Hz, 1H), 4.41 (s, 2H), 4.19 (t, J = 6.1 Hz, 2H), 2.84 (t, J = 5.8 Hz, 2H), 2.67 (s, 3H), 2.28 – 2.20 (m, 3H), 2.14 (q, J = 5.8 Hz, 1H); ¹³C NMR (100 MHz, Chloroform-d) δ 177.03, 154.82, 138.19, 133.09, 128.04, 127.29, 124.68, 123.58, 122.77, 114.02, 111.10, 80.88 (d, J_C-F = 164.0 Hz), 64.72 (d, J_C-F = 5.0 Hz), 43.65, 40.48, 32.07, 30.36 (d, J_C-F = 20.0 Hz), 20.10. HRMS (ESI) m/z [M+H]⁺ calcd for C₁₈H₂₃FNO₃: 320.1656, found: 320.1653.

3-(((2-(4-Fluorobutoxy)-4-methylnaphthalen-1-yl)methyl)amino)propanoic acid (19d). Synthesized by general procedure G from 18d. Yellow oil; yield: 80%; ¹H NMR (400 MHz, Chloroform-d) δ 7.93 (d, J = 8.5 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.36 (t, J = 7.7 Hz, 1H), 7.28 – 7.25 (m, 1H), 7.04 (s, 1H), 4.51 (t, J = 5.7 Hz, 1H), 4.40 – 4.37 (m, 3H), 4.08 (t, J = 6.5 Hz, 2H), 2.83 (t, J = 5.7 Hz, 2H), 2.63 (s, 3H), 2.26 (t, J = 5.7 Hz, 2H), 1.95 – 1.75 (m, 4H). ¹³C NMR (100 MHz, Chloroform-d) δ 177.14, 154.95, 138.16, 133.08, 127.94, 127.25, 124.58, 123.51, 122.83, 114.05, 110.75, 83.71 (d, J_C-F = 164.0 Hz), 68.19, 43.47, 40.21, 32.00, 26.96 (d, J_C-F = 19.0 Hz), 25.36 (d, J_C-F = 6.0 Hz), 20.03. HRMS (ESI) m/z [M+H]⁺ calcd for C₁₉H₂₅FNO₃: 334.1818, found: 334.1815.

3-(((2-((5-Fluoropentyl)oxy)-4-methylnaphthalen-1-yl)methyl)amino)propanoic acid (19e). Synthesized by general procedure G from 18e. White solid; yield: 80%; MP: 102−105 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.97 (d, J = 8.5 Hz, 1H), 7.88 (d, J = 8.3 Hz, 1H), 7.38 (t, J = 7.4 Hz, 1H), 7.31 (d, J = 7.7 Hz, 1H), 7.07 (s, 1H), 4.50 – 4.46 (m, 3H), 4.37 (t, J = 5.9 Hz, 1H), 4.08 (t, J = 6.6 Hz, 2H), 2.87 (t, J = 5.8 Hz, 2H), 2.67 (s, 3H), 2.31 (t, J = 5.7 Hz, 2H), 1.86 (p, J = 6.9 Hz, 2H), 1.79 – 1.65 (m, 2H), 1.57 – 1.50 (m, 2H); ¹³C NMR (100 MHz, Chloroform-d) δ 176.92, 155.05, 138.17, 133.09, 127.96, 127.30, 124.63, 123.51, 122.83, 114.08, 110.72, 83.84 (d, J_C-F = 164.0 Hz), 68.51, 43.43, 40.23, 31.82, 29.98 (d, J_C-F = 20.0 Hz), 28.83, 21.78 (d, J_C-F = 5.0 Hz), 20.10. HRMS (ESI) m/z [M+H]⁺ calcd for C₂₀H₂₇FNO₃: 348.1969, found: 348.1967.

3-(((2-((6-fluorohexyl)oxy)-4-methylnaphthalen-1-yl)methyl)amino)propanoic acid (19f). Synthesized by general procedure G from 18f. Yellow solid; yield: 81%; MP: 115−117 °C; ¹H NMR (400 MHz, Chloroform-d) δ 7.98 (d, J = 8.3 Hz, 1H), 7.88 (d, J = 8.2 Hz, 1H), 7.39 (t, J = 7.1 Hz, 1H), 7.30 (t, J = 7.4 Hz, 1H), 7.07 (s, 1H), 4.48 – 4.45 (m, 3H), 4.35 (t, J = 6.0 Hz, 1H), 4.09 (t, J = 6.7 Hz, 2H), 2.87 (t, J = 5.6 Hz, 2H), 2.67 (s, 3H), 2.35 – 2.32 (m, 2H), 1.87 – 1.82 (m, 3H), 1.73 – 1.63 (m, 2H), 1.46 – 1.44 (m, 4H); ¹³C NMR (100 MHz, Chloroform-d) δ 176.98, 155.18, 138.25, 133.15, 128.01, 127.37, 124.67, 123.55, 122.91, 114.16, 110.62, 84.02 (d, J_C-F = 163.0 Hz), 68.71, 43.30, 40.16, 31.85, 30.30 (d, J_C-F = 14.0 Hz), 29.20, 25.63, 24.99 (d, J_C-F = 5.0 Hz), 20.13. HRMS (ESI) m/z [M+H]⁺ calcd for C₂₁H₂₉FNO₃: 362.2126, found: 362.2123.

Ethyl 1-((4-bromo-2-(((trans)-4-(tert-butyl)cyclohexyl)oxy)naphthalen-1-yl)methyl)-piperidine-4-carboxylate (21). Synthesized by general procedure F from 20. Yellow oil; yield: 56%; ¹H NMR (400 MHz, Chloroform-d) δ 8.25 (dd, J = 8.6, 1.2 Hz, 1H), 8.15 (dd, J = 8.3, 1.4 Hz, 1H), 7.56 (s, 1H), 7.52 – 7.48 (m, 1H), 7.46 – 7.41 (m, 1H), 4.23 – 4.15 (m, 1H), 4.12 (q, J = 7.1 Hz, 2H), 3.88 (s, 2H), 2.90 (dt, J = 11.8, 3.7 Hz, 2H), 2.32 – 2.25 (m, 1H), 2.22 – 2.12 (m, 4H), 1.90 – 1.81 (m, 4H), 1.73 – 1.63 (m, 2H), 1.51 – 1.42 (m, 2H), 1.23 (t, J = 7.1 Hz, 3H), 1.19– 1.05 (m, 3H), 0.89 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 175.3, 135.5, 135.1, 127.7, 127.0, 126.7, 125.4, 124.7, 122.8, 121.0, 120.4, 78.6, 60.1, 52.8, 52.1, 47.3, 41.4, 32.8, 32.3, 28.3, 27.6, 25.5, 14.2. HRMS (ESI) m/z [M+H]⁺ calcd for C₂₉H₄₁BrNO₃: 530.2264, found: 530.2266.

Ethyl 1-((2-(((trans)-4-(tert-butyl)cyclohexyl)oxy)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-1-yl)methyl)piperidine-4-carboxylate (22). Synthesized by general procedure D-i) from 21. Yellow oil; yield: 69%; ¹H NMR (400 MHz, Chloroform-d) δ 8.69 (dd, J = 8.3, 1.4 Hz, 1H), 8.31 – 8.21 (m, 1H), 7.79 (s, 1H), 7.47 – 7.43 (m, 1H), 7.41 – 7.36 (m, 1H), 4.28 (tt, J = 11.0, 4.5 Hz, 1H), 4.10 (q, J = 7.2 Hz, 2H), 3.96 (s, 2H), 2.93 – 2.89 (m, 2H), 2.30 – 2.13 (m, 5H), 1.88 – 1.79 (m, 4H), 1.69 (td, J = 13.0, 12.3, 3.6 Hz, 2H), 1.43 (s, 12H), 1.23 (t, J = 7.1 Hz, 4H), 1.16 – 1.08 (m, 3H), 0.89 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 175.4, 153.1, 134.4, 132.9, 128.3, 125.8, 125.6, 125.2, 124.7, 123.9, 110.0, 83.8, 78.4, 60.1, 52.9, 52.2, 47.4, 41.4, 33.1, 32.3, 29.7, 28.3, 27.7, 25.6, 24.9, 14.2. HRMS (ESI) m/z [M+H]⁺ calcd for C₃₅H₅₃BNO₅: 578.4011, found: 578.4011.

Ethyl 1-((2-(((trans)-4-(tert-butyl)cyclohexyl)oxy)-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)naphthalen-1-yl)methyl)piperidine-4-carboxylate (23). Synthesized by general procedure M from 10. Yellow oil; yield: 45%; ¹H NMR (400 MHz, Chloroform-d) δ 7.66 (d, J = 9.0 Hz, 1H), 7.36 (s, 1H), 6.87 (s, 1H), 6.72 (d, J = 8.9 Hz, 1H), 4.25 – 4.18 (m, 1H), 4.07 (q, J = 7.2 Hz, 2H), 3.95 (s, 2H), 3.09 (d, J = 11.7 Hz, 2H), 2.60 (s, 3H), 2.38 – 2.28 (m, 3H), 2.22 – 2.18 (m, 2H), 1.91 – 1.81 (m, 6H), 1.43 (q, J = 11.8 Hz, 2H), 1.19 (t, J = 7.1 Hz, 3H), 1.16 – 1.08 (m, 3H), 0.89 (s, 9H); ¹³C NMR (100 MHz, Chloroform-d) δ 174.86, 155.01, 154.20, 136.02, 135.90, 130.22, 126.02, 123.12, 115.61, 113.47, 106.66, 77.57, 60.26, 53.15, 47.34, 33.02, 32.35, 27.65, 27.17, 25.56, 24.84, 24.56, 19.97, 14.14. HRMS (ESI) m/z [M+H]⁺ calcd for C₃₀H₄₄NO₄: 482.3265, found: 482.3261.

In vitro binding assay.

To determine the potency (IC₅₀) and selectivity of our new S1PR5 compounds toward human recombinant S1PR5 and other S1PRs, a [³²P]S1P competitive radioligand binding assays were performed using previously reported procedures. Multi-ScreenHTS FC filter plate (MilliporeSigma, Burlington, MA; CatalogNo. MSFCN6B) and ChemiSCREEN recombinant human S1PR5 membrane (MilliporeSigma, Burlington, MA, Catalog No. HTS193M) were used. All plates were blocked with a blocking solution consisting of 0.125% polyethyleneimine and 0.5% BSA at room temperature for 30 minutes and then washed three times with PBS solution. [³²P]S1P was dissolved in DMSO, and then diluted in the assay buffer (50 mM HEPES-Na pH = 7.4, 100 mM NaCl, 10 mM MgCl₂, 0.1 mM EDTA, 0.5% fatty acid-free bovine serum albumin, pH = 7.5 and protease inhibitor cocktail in deionized H₂O). All test compounds were dissolved and stored in DMSO, and serial dilutions were carried out in assay buffer. [³²P]S1P, test compounds, and S1PR5 membrane were mixed and incubated for 1 h at 30 °C with gentle shaking. The final volume of the incubation mixture was 150 μL per well. Reactions were terminated by vacuum filtration using a MultiScreenHTS vacuum manifold hooked up to a Laboport N 816 vacuum pump. Samples were washed four times using 230 μL of assay buffer. After the final wash, the filter plate was vacuum-dried overnight on the manifold. Once dried, the membranes were transferred to vials and counted on a Beckman LS3801 scintillation counter. Data was processed using GraphPad Prism and IC₅₀ values were determined using a nonlinear regression one-site competitive binding analysis. Each assay was repeated at least three times with duplicate wells for each compound. To determine compound selectivity, competitive binding assays were performed using the same procedure as described above with recombinant human S1PR1, 2, 3, and 4 membranes.

Molecular docking study methods.

Molecular docking studies were conducted to evaluate the binding interactions between small molecule ligands and the target receptor with a fork of the Smina docking suite.⁴² A semi-flexible docking approach was employed to allow for flexibility in the ligand during the docking process. The crystal structure of the receptor (PDB ID: 7YXA) was retrieved from the Protein Data Bank (http://www.rcsb.org/). Protein preparation was carried out using AutoDock Tools (http://autodock.scripps), where the structure of the protein was pre-processed by removing all water molecules and heteroatoms, and subsequently, polar hydrogens were added to optimize the structure for docking. The ligand structures were subjected to energy minimization using the MMFF94 force field to ensure geometrical stability and to obtain low-energy conformers suitable for docking. The docking simulations were performed within a 3D grid box defined around the receptor’s active site, which was centered on the centroid of the co-crystallized ligand. The dimensions of the grid box were optimized to closely encompass the ligand’s size, ensuring an accurate representation of the binding pocket. An exhaustiveness parameter of 24 was used to ensure a thorough exploration of the binding conformations. Post-docking, the binding modes of the ligands were analyzed in detail to identify key interactions with the receptor. Visualization of docking results and binding interactions was performed using PyMOL (https://pymol.org/2/) and Discovery Studio Visualizer (BIOVIA, San Diego, CA, USA), enabling the generation of high-quality images and in-depth structural analysis.

Radiochemistry.

General Information. Production of [¹¹C]CH₃I: No-carrier added [¹¹C]CO₂ was produced through Washington University, Mallinckrodt Institute of Radiology TR19 cyclotron (Advanced Cyclotron Systems, Richmond, BC, Canada) from the ¹⁴N (p,α)¹¹C nuclear reaction with proton bombardment of an aluminum target filled with 0.5% O₂ in nitrogen gas. The irradiation was achieved with a beam of 18.0 MeV at current of 40 μA for 25 min. After the irradiation was finished, the [¹¹C]CO₂ was shunted to the GE TracerLab FX MeI module to generate reactive [¹¹C]methyl iodide ([¹¹C]CH₃I) for tracer production. Briefly, up to 2400 mCi [¹¹C]CO₂ was trapped in molecular sieves and further reduced to [¹¹C]CH₄ in the presence of Ni catalyst and hydrogen at 350 °C. After removal of unreacted CO₂ and H₂O through passing a double ascarite/sicapent trap, the [¹¹C]CH₄ was concentrated and purified by trapping in a carbosphere column. Once the [¹¹C]CH₄ was released, it reacted with iodine at 740 °C to produce [¹¹C]CH₃I in a recirculation loop. Approximately 10 min following the end-of-bombardment (EOB), 800–1100 mCi of [¹¹C]CH₃I were delivered in the carrier gas to the hot cell where the radiosynthesis was accomplished.

Production of [¹⁸F]KF: The [¹⁸F]fluoride in a 0.2–2.5 mL bolus of [¹⁸O]H₂O was trapped on a pre-conditioned QMA cartridge (WAT023525, Waters) to remove [¹⁸O]H₂O and other aqueous impurities. [¹⁸F]Fluoride was eluted into the reaction vessel using aqueous potassium carbonate solution (3.0 mg/mL).

Radiosynthesis of [¹¹C]7.

The radiosynthesis of [¹¹C]7 was achieved through the boron ester precursor 22 reacting with [¹¹C]methyl iodide via Pd catalyzed cross-coupling reaction. The automated production carried out in a GE Tracerlab FX2 M methylation automated module. Before starting the production, each vial was loaded with reagents as follows. The reactor: To the reaction vessel were added 1.0 mg of tris(dibenzylideneacetone)dipalladium (0) and 1.0 mg of tri-o-tolylphosphine dissolved in anhydrous DMF (100 μL), vial #1: 5.0 N NaOH (55 μL), EtOH (55 μL); vial #2: 1.5 mg of the precursor 22, 3 μL of the NaOH (5N) and 200 μL of DMF/H₂O (9: 1); vial #3: EtOH (800 μL) and 3 N hydrochloric acid (94 μL). The [¹¹C]CH₃I was delivered to the reaction module with a helium flow rate of 20 mL/min. Upon completion of delivery, the reaction vessel was heated to 60 °C for 3 min. After trapping and oxidative addition finished, the precursor solution in vial #2 was added to the reaction vessel by N₂ flow for 20 s, followed by heating at 60 °C for another 5 min. Then, 5.0 N NaOH (55 μL), EtOH (55 μL) in vial #1 was added to the reaction vessel by N₂ flow for 20 s, followed by heating to 80 °C for another 2 min. At last, the quench reagent in vial #3 was added to the reaction vessel and the reaction mixture was loaded onto a C-18 semi-preparative column (Agilent SB-C18, 250 × 9.4 mm, 5 μm) for HPLC purification, then eluted from the column using 85% methanol in 0.1 M ammonium formate buffer (pH 4.5) HPLC mobile phase at a flow rate of 4.0 mL/min. The UV detector has a wavelength of 254 nm. A radioactive product was collected from 11.1 to 12.5 min using a glass vial prefilled with milli-Q water (40 mL), the diluted product solution was then passed through a C18 Sep-Pak light cartridge (Part No. WAT023501) with nitrogen gas assistance. The trapped product was eluted using 10% ethanol in 0.9% saline to formulate the injection dose for QC analysis and animal studies. An aliquot of the dose sample (20 μL) was injected onto the analytic HPLC system for authentication by co-injection with the cold standard compound 7, the chemical and radiochemical purity, and molar activity of the final dose were determined. The analytic HPLC system for quality control consists of a reversed-phase HPLC analytical column (Phenomenex Prodigy, 4.6 mm × 250), UV wavelength of 254 nm, mobile phase of methanol/0.1 M ammonium formate buffer (v/v, 95/5, pH = 4.5) with the flow rate of 1.0 mL/min. Under this condition, [¹¹C]7 has a retention time of TR = 5.3 min, 11–14% radiochemical yield (decay corrected to EOB), >40.7 GBq/μmol molar activity (decay corrected to EOB), >95% chemical purity and >99% radiochemical purity.

Radiosynthesis of [¹⁸F]7a.

[¹⁸F]KF (~7.4 GBq) aqueous was added to a vial containing Kryptofix 222 (7−9 mg), and dried by a zeotropic evaporation with acetonitrile (3 × 1 mL) under nitrogen flow at 100 °C. To the reaction vial was added Cu(OTf)₂(impy)₄ (9.0 mg) and boron ester precursor 22 (4−5 mg) as a solution in N,N-dimethylacetamide (200 μL) and 1-butanol (100 μL). Then the reaction vial was sealed and heated at 110 °C for 20 min using an oil bath, agitating the reaction vessel five times during the heating period. The reaction vial was raised above the oil bath; the reaction was quenched in 3.0 mL of solvent (methanol/ammonium formate solution 80/20, pH = ~4.5); and extracted with anhydrous ether (3 × 1.5 mL), Then the reaction vial dried under nitrogen flow at room temperature. To the reaction vial was added NaOH solution (5N, 55 μL) in ethanol (200 μL). Then the reaction vial was sealed and heated again at 100 °C for 5 min using an oil bath, agitating the reaction vessel twice during the heating period. Then, the reaction was removed from the oil-bath and quenched with 3 N hydrochloric acid (94 μL) and diluted with 3.0 mL of the HPLC mobile phase (80% methanol in 0.1 M ammonium formate buffer, pH 4.5) and loaded onto a C18 column (Agilent SB-C18, 250 × 9.4 mm, 5 μm), then eluted from the column using above-mentioned HPLC mobile phase at a flow rate of 4.0 mL/min. The UV detector has a wavelength of 254 nm. A 100 mL glass vial containing 50 mL of sterile water was used to collect the radioactive product [¹⁸F]7a which was eluted from 15.2 to 19.3 min; the diluted product was then passed through a C18 Sep-Pak Plus cartridge (Part No. WAT020515) with nitrogen gas assistance. The trapped product was eluted using 10% ethanol in 0.9% saline to formulate the injection dose for QC analysis and animal studies. An aliquot of the dose sample (20 μL) was injected onto the analytic HPLC system for authentication by co-injection with the cold standard compound 7a, the chemical and radiochemical purity, and molar activity of the final dose were determined. The analytic HPLC system for quality control consists of a reversed-phase HPLC analytical column (Phenomenex Prodigy, 4.6 mm × 250), UV wavelength of 280 nm, mobile phase of methanol/0.1 M ammonium formate buffer (v/v, 92/8, pH = 4.5) with the flow rate of 1.0 mL/min. Under this condition, [¹⁸F]7a has a retention time of TR = 4.7 min, 22–29% radiochemical yield (decay corrected to EOS), >55.6 GBq/μmol molar activity (decay corrected to EOS), >95% chemical purity and >99% radiochemical purity.

Radiosynthesis of [¹¹C]12a.

The radiosynthesis of [¹¹C]12a was achieved through the naphthol precursor 23 reacting with [¹¹C]methyl iodide via N-methylation. The automated production was carried out in a GE Tracerlab FX2 M methylation automated module. Before starting the production, each vial was loaded with reagents as follows. The reactor: To the reaction vessel were added 1.5 mg of the precursor 23, 2–3 μL of the NaOH (5N) and 300 μL of anhydrous dimethylsulfoxide; vial #2: 2.5 N NaOH (90 μL); vial #3: 85% methanol in 0.1 M ammonium formate buffer, pH 4.5 (900 μL) and 3 N hydrochloric acid (80 μL). The [¹¹C]CH₃I was delivered to the reaction module with a helium flow rate of 20 mL/min. Upon completion of delivery, the reaction vessel was heated to 60 °C for 5 min. Then, 2.5 N NaOH in vial #2 was added to the reaction vessel by N₂ flow for 20 s, followed by heating to 80 °C for another 3 min. At last, the quench reagent in vial #3 was added to the reaction vessel and the reaction mixture was loaded onto a C-18 semi-preparative column (Agilent SB- C18, 250 × 9.4 mm, 5 μm) for HPLC purification, then eluted from the column using 85% methanol in 0.1 M ammonium formate buffer (pH 4.5) HPLC mobile phase at a flow rate of 4.0 mL/min. The UV detector has a wavelength of 254 nm. A radioactive product was collected from 13.5 to 15.5 min using a glass vial prefilled with milli-Q water (40 mL), the diluted product solution was then passed through a C18 Sep-Pak light cartridge (Part No. WAT023501) with nitrogen gas assistance. The trapped product was eluted using 10% ethanol in 0.9% saline to formulate the injection dose for QC analysis and animal studies. An aliquot of the dose sample (20 μL) was injected onto the analytic HPLC system for authentication by co-injection with the cold standard compound 12a, the chemical and radiochemical purity, and molar activity of the final dose were determined. The analytic HPLC system for quality control consists of a reversed-phase HPLC analytical column (Phenomenex Prodigy, 4.6 mm × 250), UV wavelength of 254 nm, mobile phase of methanol/0.1 M ammonium formate buffer (v/v, 95/5, pH = 4.5) with the flow rate of 1.0 mL/min. Under this condition, [¹¹C]12a has a retention time of TR = 4.3 min, 11–14% radiochemical yield (decay corrected to EOB), >54 GBq/μmol molar activity (decay corrected to EOB), >90% chemical purity and >99% radiochemical purity.

Radiosynthesis of [¹⁸F]12b.

Two-Pot Procedure. The intermediate 1-[¹⁸F]fluoro-2-tosyloxyethane ([¹⁸F]25) was radiosynthesized by following the published procedure⁴³. A solution of precursor 23 (1.0 mg) and NaOH (3 μL) in DMSO (300 mL) was added to the reaction vessel containing [¹⁸F]25. The vessel was capped and heated at 65 °C for 10 min. Subsequently, the reaction mixture was added NaOH solution (5N, 55 μL) in ethanol (200 μL). Then the reaction vial was sealed and heated again at 85 °C for 5 min using an oil bath, agitating the reaction vessel twice during the heating period. Then, the reaction was removed from the oil-bath and quenched with 3 N hydrochloric acid (95 μL) and diluted with 3.0 mL of the HPLC mobile phase (81% methanol in 0.1 M ammonium formate buffer, pH 4.5) and loaded onto a C18 column (Agilent SB-C18, 250 × 9.6 mm, 5 μm), then eluted from the column using above-mentioned HPLC mobile phase at a flow rate of 4.0 mL/min. The UV detector has a wavelength of 254 nm. A 100 mL glass vial containing 50 mL of sterile water was used to collect the radioactive product [¹⁸F]12b which was eluted from 17.5 to 20.5 min; the diluted product was then passed through a C18 Sep-Pak Plus cartridge (Part No. WAT020515) with nitrogen gas assistance. The trapped product was eluted using 10% ethanol in 0.9% saline to formulate the injection dose for QC analysis and animal studies. An aliquot of the dose sample (20 μL) was injected onto the analytic HPLC system for authentication by co-injection with the cold standard compound 12b, the chemical and radiochemical purity, and molar activity of the final dose were determined. The analytic HPLC system for quality control consists of a reversed-phase HPLC analytical column (Phenomenex Prodigy, 4.6 mm × 250), UV wavelength of 280 nm, mobile phase of methanol/0.1 M ammonium formate buffer (v/v, 95/5, pH = 4.5) with the flow rate of 1.0 mL/min. Under this condition, [¹⁸F]7a has a retention time of TR = 3.8 min, 16–25% radiochemical yield (decay corrected to EOS), >50 GBq/μmol molar activity (decay corrected to EOS), >90% chemical purity and >99% radiochemical purity.

Ex vivo biodistribution study in rodents.

To investigate the tissue distribution and kinetics of [¹¹C]7, [¹⁸F]7a, [¹¹C]12a, and [¹⁸F]12b, and in living animals, the ex vivo biodistribution study was performed using male adult Sprague Dawley (SD) rats. For [¹¹C]7, approximately 3.7 MBq of [¹¹C]7 in 100 μL of buffer was injected via the tail vein into SD rats (male; 7–8 weeks old; 240–260 g). Rats were euthanized under 2–3% isoflurane/oxygen anesthesia at 5, 30, and 60 min post-injection (n = 3 per group). For [¹⁸F]7a, approximately 1.48 MBq of [¹⁸F]7a in 100 μL of buffer was injected via the tail vein into SD rats (male; 7–8 weeks old; 200–220 g). Rats were euthanized under 2–3% isoflurane/oxygen anesthesia at 5, 30, 60 and 120 min post-injection (n = 4 per group). For [¹¹C]12a, approximately 3.7 MBq of [¹¹C]12a in 100 μL of buffer was injected via the tail vein into SD rats (male; 9–10 weeks old; 260–300 g). Rats were euthanized under 2–3% isoflurane/oxygen anesthesia at 5, 30, and 60 min post-injection (n = 3 per group). For [¹⁸F]12b, approximately 1.48 MBq of [¹⁸F]12b in 100 μL of buffer was injected via the tail vein into SD rats (male; 7–8 weeks old; 200–220 g). Rats were euthanized under 2–3% isoflurane/oxygen anesthesia at 5, 30, and 60 post-injection (n = 4 per group). Tissues of interest including blood, lung, liver, spleen, kidney, muscle, fat, heart, pancreas, brain, brain stem, and tail were collected, weighed, and counted on a Beckman 8000 automated gamma counter (Beckman, Brea, CA), with a standard dilution of the injected dose. The uptake of each organ was decay corrected, calculated and expressed as a percentage of the injection dose per gram of wet tissue (%ID per gram).

K_d and Bmax value determination.

Saturation binding assay and saturation autoradiography with [¹⁸F]7a were performed for recombinant human S1PR5 membrane, mouse brain tissue, and postmortem human cerebral cortex. For K_d determination on human S1PR5 membrane, saturation binding affinity assay was performed with a serial dilution of [¹⁸F]7a ranging from 0.02 to 33 nM. Dilutions of [¹⁸F]7a were incubated with S1PR5 membrane and processed similarly to the competitive binding assay and counted accordingly. The non-specific binding was determined by incubation in the presence of 100 μM compound 7. For K_d determination in mouse brain tissue and postmortem human cerebral cortex tissues, 20 μm fresh frozen mice brain tissue sections were prewarmed and pre-incubated with HBSS buffer containing 10 mM HEPES, 5 mM MgCl₂, 1 mM CaCl₂, and 0.5 % BSA, and 0.1 mM EDTA at pH 7.4 for 10 min. The sections were then incubated with a serial dilution of [¹⁸F]7a ranging from 6.25 to 800 nM for 45 min, and then washed, and air dried with a blower. Dried slides were incubated with a BAS-IP MS Storage Phosphor Screen (Cytiva, Marlborough, MA) overnight. Autoradiography signal was then visualized using a Typhoon FLA 9000 laser scanner (Fiji Photo Film, Tokyo, Japan). Images were then quantified using Multi Gauge v3.0 software (Fiji Photo Film, Tokyo, Japan). The non-specific binding was determined by incubation in the presence of 100 μM compound 7. All K_d was determined using a nonlinear regression one-site saturation binding fit model in GraphPad Prism.

Preparation of blocking reagent solutions and PET imaging of [¹⁸F]7a in C57BL/6 mice with blocking.

For in vivo studies with injection of blocking agent to animals: 4 mg of the blocking agent was weighed into a 6 mL glass vial. To this, 0.2 mL of Polyethylene Glycol 300, 0.6 mL of a 45% (w/v) 2-hydroxypropyl beta-cyclodextrin solution (prepared by dissolving 9.0 g in 16 mL of water), and 1.2 mL of saline solution (0.9%) were added. The mixture was vortexed for 2 minutes to ensure initial mixing, followed by sonication until the blocking agent was fully dissolved, resulting in a clear and transparent solution. This prepared solution ensured optimal solubility and uniformity for experimental applications. Blocking agent was injected intravenously 5 min prior to the injection of radiotracers at a dose of 4 mg/kg.

MicroPET scan in control mice and mice with pretreatment of S1PR5 modulator were performed using a Mediso PET/CT system under 1.5–2% isoflurane/oxygen inhalation anesthesia, with the animal placed in transaxial position and centered with the T13 vertebral spine in the field of view. Following a CT scan for anatomical co-registration, each mouse received a bolus intravenous injection of the [¹⁸F]7a (15–22 MBq). A list mode protocol (1 × 3, 6 × 2, 9 × 5, 6 × 10, 4 × 30, 2 × 60 s, 2 × 2, 10 × 5 min) was used with 60-min dynamic data acquisition. The reconstructed PET image was analyzed using PMOD (version 4.301, Bruker, Switzerland). The Mouse Brain (Waxholm-40um) brain template (https://www.nitrc.org/projects/incfwhsmouse) was normalized to the individual mouse brain in the CT images, with the corresponding atlas to obtain the regional brain uptakes. The radioligand uptake was normalized by body weight and injection dose to obtain the standard uptake value (SUV). SUV values of cuprizone mice vs control mice was compared using independent one-way t-test. A p-value < 0.05 was considered statistically significant.

Induction of C57BL/6 cuprizone mice and PET imaging study of [¹⁸F]7a in cuprizone model of MS.

Female C57BL/6J mice (strain 000664) were purchased from Jackson Laboratories (Bar Harbor, ME, USA) and kept in normal day–night cycle (12/12) with ad libitum access to food and water. At eight weeks old, mice were randomly separated in two groups: a control group (n = 4 mice) that was fed a standard rodent chow for the entire experimental period, and a cuprizone group (n = 4 mice) in which mice received a 0.2% cuprizone (Sigma-Aldrich) diet for four weeks to induce cerebral demyelination and inflammation. All experimental procedures were approved by the Institutional Animal Care and Use Committee of Washington University in St. Louis. MicroPET scans in cuprizone and control mice (n = 4 per group) were performed using a Mediso PET/CT system, similarly as described above for baseline and blocking studies.

MicroPET brain imaging study of [¹⁸F]7a in macaque.

To check if [¹⁸F]7a enter the brain of nonhuman primates (NHP), PET brain imaging study was performed using an MRS*PET/CT 220 scanner (MR Solutions, Guildford, United Kingdom) for data acquisition. A male macaque was scanned under anesthetization (induced with ketamine and glycopyrrolate and maintained with the inhalation of isoflurane combined with nitrous and oxygen). Core temperature was kept at 37 °C with a heated Bair Hugger warming system. Dynamic PET scans were collected from 0 to 120 min after intravenous administration of ~158 MBq of the adiotracer with the following time frames: 3 × 1 min, 4 × 2 min, 3 × 3 min, and 20 × 5 min. Emission data were corrected for dead time, scatter, random, randoms, attenuation, with normalizationand then reconstructed using ordered subset expectation maximization (OSEM) with 4 iterations and 16 subsets. For quantitative analyses, dynamic PET images were motion-corrected and registered to a standardized monkey MRI template and atlas (Rhesus Macaque NIA19 Brain, https://www.nitrc.org/projects/inia19/) using an in-house pipeline written in MATLAB (R2024a, The Mathworks Inc., USA). Predefined brain regions of interest from the atlas were used to obtain regional time-activity curves, which were normalized by body weight and the injected dose to obtain standardized uptake values (SUVs).

In vitro autoradiography and immunohistology study in cuprizone and control mouse brain.

Autoradiography analysis of [¹⁸F]7a was performed on control and cuprizone-treated mouse brains similarly as described in saturation autoradiography analysis. Briefly, 10 μm brain sections were prewarmed and preincubated with buffer, and then incubated with ~370 kBq of [¹⁸F]7a per slide for 1 hour with gentle shaking, washed three times in buffer and dipped in ice-cold water, and then dried. Dried slides were incubated with a BAS-IP MS Storage Phosphor Screen (Cytiva, Marlborough, MA) and visualized using a Typhoon FLA 9000 laser scanner (Fiji Photo Film, Tokyo, Japan). Images were then quantified using Multi Gauge v3.0 software (Fiji Photo Film, Tokyo, Japan). The non-specific binding was determined by incubation in the presence of 100 μM compound 7.

Immunofluorescent analysis of S1PR5 was performed in adjacent tissue slides. Sections were fixed with 4% paraformaldehyde for 10 min and washed 3 times for 5 min each. Sections were then blocked and permeabilized using 5% horse serum with 0.15% Triton X-100 for 30 min, washed, and incubated with primary antibody overnight at 4 °C. Antibodies used were S1PR5 antibody for S1PR5 expression (Novus, catalog: 24712); Olig2 for oligodendrocytes (R&D systems, catalog: AF2418); MBP for myelin structure (Santa Cruz, catalog: sc-271524). Sections were then washed and incubated with Alexa fluor secondary antibodies (Thermofisher) at room temperature for 1 hour, washed three times, and mounted using EverBrite^™ Mounting Medium with DAPI (Biotium). Whole slide scan was performed using ZEISS Axioscan 7 slide scanner. Images were processed and analyzed using QuPath, and positively stained cells were counted.

Radiometabolite analysis.

To characterize the profiles of the radiotracer, HPLC radiometabolite analysis was conducted for [¹⁸F]7a using animal plasma or rat brain tissue samples collected at different time points post-injection.

Radiometabolite analysis of [¹⁸F]7a in rats.

SD rats (~200 g) were intravenously injected with ~44.4 MBq of [¹⁸F]7a. Rats were euthanized and heparinized blood (~6 mL) and brain were collected at 5, 30, 60, and 90 minutes post-injection. Plasma was separated by centrifugation and mixed with solid urea to solubilize plasma proteins, enabling back-flushing of parent compounds and metabolites from the capture column to the analytical column. The plasma-urea mixture (final concentration 8 M urea, ~5 mL) was filtered with a Whatman^® GD/X syringe filter (1 μm pore size, 13 mm diameter) and injected into a 5 mL HPLC loop. The capture column was eluted with 1% acetonitrile in ammonium formate (0.1 M, pH = 4.5) at a flow rate of 2 mL/min for 4 minutes. Radioactivity enriched in the capture column was reversely eluted into an analytical C18 column. For brain radiometabolite analysis, total brain samples at each time point were collected, homogenized manually in ice-cold PBS using a Dounce homogenizer, and solubilized using urea to achieve a final concentration of 8 M in ~4 mL. The homogenate was centrifuged, and the supernatant was collected and filtered through a syringe filter for HPLC analysis, a mobile phase of 55% acetonitrile in 0.1 M ammonium formate buffer, pH = 4.2 was used; and a flow rate was at 1.0 mL min⁻¹.

Radiometabolite analysis of [¹⁸F]7a in Macaques.

HPLC radiometabolite analysis of the macaque plasma samples collected at different time points post-injection of [¹⁸F]7a was in conjunction with PET imaging performed as previously reported.^44–46. A male macaque (~10 kg) was intravenously injected with ~0.13 GBq of [¹⁸F]7a. Arterial blood samples (~7 mL) were collected at 5-, 30-, 60-, and 90-minutes post-injection. The remaining steps followed the same procedure as described for [¹⁸F]7a in rats.

Supplementary Material

S1P5 supplementary

NIHMS2179144-supplement-S1P5_supplementary.pdf^{(11.5MB, pdf)}

Supporting information is available free of charge via the Internet at http://pubs.acs.org., which includes Synthesis of compound 8, optimization of reaction conditions for C-11 labeling of [¹¹C]7, SUV of rodent brain uptake for cuprizone-induced mouse model of MS and control mouse, radiochemistry HPLC chromatograms, compound purity, NMR spectra of compounds; and reference, Molecular formula strings for final compounds (CSV).

ACKNOWLEDGMENT

We would like to thank the Washington University High-Resolution NMR Facility for assistance in characterizing our compounds, the Cyclotron staff for their assistance in providing [¹⁸F]fluoride and [¹¹C]methyl iodine, Linlin Tian for their assistance with the NHP imaging studies. We also thank Washington University small animal PET Imaging Facility Nikki Fettig, Margaret Morris, and Emily Williams for their assistants on the rodent study.

Funding Sources

This work was supported by the National Institutes of Health including the National Institute of Neurological Disorders and Stroke (NINDS) and National Institute of Aging (NIA) [NS103988, NS134586, NS75527, NS0103957], and the National Institute of Biomedical Imaging and Bioengineering [EB025815], Barnes Jewish Foundation, American Parkinson Disease Association (APDA), the Missouri Chapter of the APDA, and the Paula & Rodger Riney Fund.

ABBREVIATIONS

BBB: blood–brain barrier
CH₃I: methyl iodine
CNS: central nervous system
DCM: dichloromethane
DIPEA: N,N-diisopropylethylamine
DMF: dimethylformamide
EtOAc: ethyl acetate
HPLC: High-performance liquid chromatography
MeCN: Acetonitrile
MRI: Magnetic resonance imaging
MS: multiple sclerosis
NaOH: Sodium hydroxide
NFSI: N-fluorobenzenesulfonimide
NHP: nonhuman primates
PET: positron emission tomography
PMOD: Peripheral module interface
QC: quality control
RT: room temperature
S1PR5: sphingosine-1-phosphate receptor 5
SD: sprague dawley
SUV: standardized uptake value
TAC: time-activity curve
THF: tetrahydrofuran
TLC: thin layer chromatography
%ID/g: percentile injection dose per gram

Footnotes

The authors declare no conflict of financial interest.

REFERENCES

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1P5 supplementary

NIHMS2179144-supplement-S1P5_supplementary.pdf^{(11.5MB, pdf)}

[R1] (1).An S; Goetzl EJ; Lee H Signaling mechanisms and molecular characteristics of G protein-coupled receptors for lysophosphatidic acid and sphingosine 1-phosphate. Journal of Cellular Biochemistry 1998, 72 (S30–31), 147–157. [DOI] [PubMed] [Google Scholar]

[R2] (2).Kluk MJ; Hla T Signaling of sphingosine-1-phosphate via the S1P/EDG-family of G-protein-coupled receptors. Biochim Biophys Acta 2002, 1582 (1–3), 72–80. [DOI] [PubMed] [Google Scholar]

[R3] (3).Van Brocklyn JR; Lee MJ; Menzeleev R; Olivera A; Edsall L; Cuvillier O; Thomas DM; Coopman PJ; Thangada S; Liu CH; et al. Dual actions of sphingosine-1-phosphate: extracellular through the Gi-coupled receptor Edg-1 and intracellular to regulate proliferation and survival. J Cell Biol 1998, 142 (1), 229–240. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R4] (4).Hla T Signaling and biological actions of sphingosine 1-phosphate. Pharmacol Res 2003, 47 (5), 401–407. [DOI] [PubMed] [Google Scholar]

[R5] (5).Kono M; Allende ML; Proia RL Sphingosine-1-phosphate regulation of mammalian development. Biochim Biophys Acta 2008, 1781 (9), 435–441. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R6] (6).LaMontagne K; Littlewood-Evans A; Schnell C; O’Reilly T; Wyder L; Sanchez T; Probst B; Butler J; Wood A; Liau G; et al. Antagonism of sphingosine-1-phosphate receptors by FTY720 inhibits angiogenesis and tumor vascularization. Cancer Res 2006, 66 (1), 221–231. [DOI] [PubMed] [Google Scholar]

[R7] (7).Maceyka M; Harikumar KB; Milstien S; Spiegel S Sphingosine-1-phosphate signaling and its role in disease. Trends Cell Biol 2012, 22 (1), 50–60. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R8] (8).Brinkmann V Sphingosine 1-phosphate receptors in health and disease: mechanistic insights from gene deletion studies and reverse pharmacology. Pharmacol Ther 2007, 115 (1), 84–105. [DOI] [PubMed] [Google Scholar]

[R9] (9).Blaho VA; Hla T An update on the biology of sphingosine 1-phosphate receptors. J Lipid Res 2014, 55 (8), 1596–1608. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R10] (10).Chun J; Giovannoni G; Hunter SF Sphingosine 1-phosphate Receptor Modulator Therapy for Multiple Sclerosis: Differential Downstream Receptor Signalling and Clinical Profile Effects. Drugs 2021, 81 (2), 207–231. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R11] (11).Hobson AD; Harris CM; van der Kam EL; Turner SC; Abibi A; Aguirre AL; Bousquet P; Kebede T; Konopacki DB; Gintant G; et al. Discovery of A-971432, An Orally Bioavailable Selective Sphingosine-1-Phosphate Receptor 5 (S1P5) Agonist for the Potential Treatment of Neurodegenerative Disorders. J Med Chem 2015, 58 (23), 9154–9170. [DOI] [PubMed] [Google Scholar]

[R12] (12).Schmidt KG; Herrero San Juan M; Trautmann S; Berninger L; Schwiebs A; Ottenlinger FM; Thomas D; Zaucke F; Pfeilschifter JM; Radeke HH Sphingosine-1-Phosphate Receptor 5 Modulates Early-Stage Processes during Fibrogenesis in a Mouse Model of Systemic Sclerosis: A Pilot Study. Front Immunol 2017, 8, 1242. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R13] (13).Shah MV; Zhang R; Irby R; Kothapalli R; Liu X; Arrington T; Frank B; Lee NH; Loughran TP Jr. Molecular profiling of LGL leukemia reveals role of sphingolipid signaling in survival of cytotoxic lymphocytes. Blood 2008, 112 (3), 770–781. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R14] (14).Kim HJ; Miron VE; Dukala D; Proia RL; Ludwin SK; Traka M; Antel JP; Soliven B Neurobiological effects of sphingosine 1-phosphate receptor modulation in the cuprizone model. FASEB J 2011, 25 (5), 1509–1518. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R15] (15).Snaidero N; Simons M Myelination at a glance. J Cell Sci 2014, 127 (Pt 14), 2999–3004. [DOI] [PubMed] [Google Scholar]

[R16] (16).Jaillard C; Harrison S; Stankoff B; Aigrot MS; Calver AR; Duddy G; Walsh FS; Pangalos MN; Arimura N; Kaibuchi K; et al. Edg8/S1P5: an oligodendroglial receptor with dual function on process retraction and cell survival. J Neurosci 2005, 25 (6), 1459–1469. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R17] (17).Zirngibl M; Assinck P; Sizov A; Caprariello AV; Plemel JR Oligodendrocyte death and myelin loss in the cuprizone model: an updated overview of the intrinsic and extrinsic causes of cuprizone demyelination. Mol Neurodegener 2022, 17 (1), 34. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R18] (18).Praet J; Guglielmetti C; Berneman Z; Van der Linden A; Ponsaerts P Cellular and molecular neuropathology of the cuprizone mouse model: clinical relevance for multiple sclerosis. Neurosci Biobehav Rev 2014, 47, 485–505. [DOI] [PubMed] [Google Scholar]

[R19] (19).van Doorn R; Lopes Pinheiro MA; Kooij G; Lakeman K; van het Hof B; van der Pol SM; Geerts D; van Horssen J; van der Valk P; van der Kam E; et al. Sphingosine 1-phosphate receptor 5 mediates the immune quiescence of the human brain endothelial barrier. J Neuroinflammation 2012, 9, 133. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R20] (20).O’Sullivan C; Schubart A; Mir AK; Dev KK The dual S1PR1/S1PR5 drug BAF312 (Siponimod) attenuates demyelination in organotypic slice cultures. J Neuroinflammation 2016, 13, 31. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R21] (21).Uchi T; Konno S; Kihara H; Fujioka T Siponimod ameliorates experimental autoimmune neuritis. J Neuroinflammation 2023, 20 (1), 35. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R22] (22).Cohan S; Chen C Sphingosine-1-phosphate, its receptors, and their role in multiple sclerosis. In Translational Neuroimmunology, Volume 8, 2023; pp 145–173. [Google Scholar]

[R23] (23).Perez-Jeldres T; Tyler CJ; Boyer JD; Karuppuchamy T; Yarur A; Giles DA; Yeasmin S; Lundborg L; Sandborn WJ; Patel DR; et al. Targeting Cytokine Signaling and Lymphocyte Traffic via Small Molecules in Inflammatory Bowel Disease: JAK Inhibitors and S1PR Agonists. Front Pharmacol 2019, 10, 212. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R24] (24).Kurata H; Kusumi K; Otsuki K; Suzuki R; Kurono M; Komiya T; Hagiya H; Mizuno H; Shioya H; Ono T; et al. Discovery of a 1-Methyl-3,4-dihydronaphthalene-Based Sphingosine-1-Phosphate (S1P) Receptor Agonist Ceralifimod (ONO-4641). A S1P(1) and S1P(5) Selective Agonist for the Treatment of Autoimmune Diseases. J Med Chem 2017, 60 (23), 9508–9530. [DOI] [PubMed] [Google Scholar]

[R25] (25).Walzer T; Chiossone L; Chaix J; Calver A; Carozzo C; Garrigue-Antar L; Jacques Y; Baratin M; Tomasello E; Vivier E Natural killer cell trafficking in vivo requires a dedicated sphingosine 1-phosphate receptor. Nat Immunol 2007, 8 (12), 1337–1344. [DOI] [PubMed] [Google Scholar]

[R26] (26).Mandala S; Hajdu R; Bergstrom J; Quackenbush E; Xie J; Milligan J; Thornton R; Shei GJ; Card D; Keohane C; et al. Alteration of lymphocyte trafficking by sphingosine-1-phosphate receptor agonists. Science 2002, 296 (5566), 346–349. [DOI] [PubMed] [Google Scholar]

[R27] (27).Matloubian M; Lo CG; Cinamon G; Lesneski MJ; Xu Y; Brinkmann V; Allende ML; Proia RL; Cyster JG Lymphocyte egress from thymus and peripheral lymphoid organs is dependent on S1P receptor 1. Nature 2004, 427 (6972), 355–360. [DOI] [PubMed] [Google Scholar]

[R28] (28).Lamb YN Ozanimod: First Approval. Drugs 2020, 80 (8), 841–848. [DOI] [PubMed] [Google Scholar]

[R29] (29).Miron VE; Jung CG; Kim HJ; Kennedy TE; Soliven B; Antel JP FTY720 modulates human oligodendrocyte progenitor process extension and survival. Ann Neurol 2008, 63 (1), 61–71. [DOI] [PubMed] [Google Scholar]

[R30] (30).Scott FL; Clemons B; Brooks J; Brahmachary E; Powell R; Dedman H; Desale HG; Timony GA; Martinborough E; Rosen H; et al. Ozanimod (RPC1063) is a potent sphingosine-1-phosphate receptor-1 (S1P1 ) and receptor-5 (S1P5 ) agonist with autoimmune disease-modifying activity. Br J Pharmacol 2016, 173 (11), 1778–1792. [DOI] [PMC free article] [PubMed] [Google Scholar]

[R31] (31).Lyapina E; Marin E; Gusach A; Orekhov P; Gerasimov A; Luginina A; Vakhrameev D; Ergasheva M; Kovaleva M; Khusainov G; et al. Structural basis for receptor selectivity and inverse agonism in S1P(5) receptors. Nat Commun 2022, 13 (1), 4736. [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Design and Synthesis of Novel PET Radiotracers for Imaging Sphingosine-1-Phosphate Receptor 5 (S1PR5) in the Brain

Jiawen Lang

Wenjuan Zhou

Tianyu Huang

Hao Jiang

Hong Chen

Shuxian Xu

Lin Qiu

Madison Heady

Yanbo Yu

Matthew R Brier

Ying-Hwey Nai

Caroline Guglielmetti

Joel S Perlmutter

Zhude Tu

Abstract

INTRODUCTION

Figure 1.

Table 2.

RESULTS AND DISCUSSION

Chemistry:

Figure 2.

Scheme 1.

Scheme 2 A&B.

Scheme 3 A-B.

In vitro determination of binding activity.

Table 1.

Molecular Docking Studies.

Figure 3.

Radiochemistry.

Scheme 4.

Scheme 5.

Ex vivo biodistribution study of [11C]7, [18F]7a, [11C]12a and [18F]12b in Sprague-Dawley rats.

Table 3.

In vitro direct binding determination of Kd and Bmax values of [18F]7a.

Figure 4.

PET/CT imaging studies in the C57BL/6 mice at baseline and pretreatment with S1PR5 modulators.

Figure 5.

PET/CT imaging in cuprizone-diet mouse model of MS and control mice.

Figure 6.

In vitro autoradiography of [18F]7a and immunohistology of S1PR5 studies in the cuprizone mouse model of MS.

Figure 7.

PET brain imaging studies in nonhuman primates (NHP).

Figure 8.

Radiometabolite analysis of [18F]7a in rat and nonhuman primates (NHP).

Figure 9.

CONCLUSION

EXPERIMENTAL SECTION

Animal Studies.

General Section:

General procedure A: Synthesis of compound 3.

General procedure B: Synthesis of compound 4.

General procedure C: Synthesis of compound 4a.

General procedure D: Synthesis of compound 4b.

General procedure E: Synthesis of compound 5, 5a and 5b.

General procedure F: Synthesis of compound 6, 6a and 6b.

General procedure G: Synthesis of compound 7, 7a and 7b.

General procedure H: Synthesis of compound 9.

General procedure I: Synthesis of compound 10.

General procedure J: Synthesis of compound 11a.

General procedure K: Synthesis of compound 11b.

General procedure L: Synthesis of compound 12a and 12b.

General procedure M: Synthesis of compound 15.

General procedure N: Synthesis of compound 16a-b.

General procedure O: Synthesis of compound 16c.

General procedure P: Synthesis of compound 16d-f.

General procedure Q: Synthesis of compound 16g-i & m.

General procedure R: Synthesis of compound 16j.

General procedure S: Synthesis of compound 16k-l, 16n and 18a-f.

General procedure T: Synthesis of compound 16o and 16p.

In vitro binding assay.

Molecular docking study methods.

Radiochemistry.

Radiosynthesis of [11C]7.

Radiosynthesis of [18F]7a.

Radiosynthesis of [11C]12a.

Radiosynthesis of [18F]12b.

Ex vivo biodistribution study in rodents.

Kd and Bmax value determination.

Ex vivo biodistribution study of [¹¹C]7, [¹⁸F]7a, [¹¹C]12a and [¹⁸F]12b in Sprague-Dawley rats.

In vitro direct binding determination of K_d and B_max values of [¹⁸F]7a.

In vitro autoradiography of [¹⁸F]7a and immunohistology of S1PR5 studies in the cuprizone mouse model of MS.

Radiometabolite analysis of [¹⁸F]7a in rat and nonhuman primates (NHP).

Radiosynthesis of [¹¹C]7.

Radiosynthesis of [¹⁸F]7a.

Radiosynthesis of [¹¹C]12a.

Radiosynthesis of [¹⁸F]12b.

K_d and Bmax value determination.

Preparation of blocking reagent solutions and PET imaging of [¹⁸F]7a in C57BL/6 mice with blocking.

Induction of C57BL/6 cuprizone mice and PET imaging study of [¹⁸F]7a in cuprizone model of MS.

MicroPET brain imaging study of [¹⁸F]7a in macaque.

Radiometabolite analysis of [¹⁸F]7a in rats.

Radiometabolite analysis of [¹⁸F]7a in Macaques.