Syntheses and in vitro evaluation of new S1PR1 compounds and initial evaluation of a lead F-18 radiotracer in rodents

Article abstract of DOI:10.1016/j.ejmech.2018.03.035

Thirteen new sphingosine-1-phosphate receptor 1 (S1PR1) ligands were designed and synthesized by replacing azetidine-3-carboxylic acid moiety of compound 4 with new polar groups. The in vitro binding potency of these new analogs toward S1PR1 was determined. Out of 13 new compounds, four compounds 9a, 10c, 12b, and 16b displayed high S1PR1 binding potency with IC₅₀ values of 13.2 ± 3.2, 14.7 ± 1.7, 9.7 ± 1.6, and 6.3 ± 1.3 nM, respectively; further binding studies of these four ligands toward S1PR2-5 suggested they are highly selective for S1PR1 over other S1PRs. The radiosynthesis of the lead radiotracer [¹⁸F]12b was achieved with good radiochemical yield (～14.1%), high radiochemical purity (>98%), and good specific activity (～54.1 GBq/μmol, decay corrected to the end of synthesis, EOS). Ex vivo autoradiography and initial biodistribution studies in rodents were performed, suggesting that [¹⁸F]12b was able to penetrate the blood-brain barrier (BBB) with high brain uptake (0.71% ID/g at 60 min post-injection) and no defluorination was observed. In vitro autoradiography study in brain slices of lipopolysaccharides (LPS)-induced neuroinflammation mice indicated that SEW2871, a specific S1PR1 ligand was able to reduce the uptake of [¹⁸F]12b, suggesting [¹⁸F]12b has S1PR1 specific binding. These initial results suggested that [¹⁸F]12b has potential to be an F-18 labeled radiotracer for imaging S1PR1 in the brain of the animal in vivo.

Full text of DOI:10.1016/j.ejmech.2018.03.035

Accepted Manuscript
Syntheses and in vitro evaluation of new S1PR1 compounds and initial evaluation of
a lead F-18 radiotracer in rodents
Zonghua Luo, Adam J. Rosenberg, Hui Liu, Junbin Han, Zhude Tu
PII:
S0223-5234(18)30278-2
10.1016/j.ejmech.2018.03.035
EJMECH 10300
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 8 January 2018
Revised Date: 27 February 2018
Accepted Date: 12 March 2018
Please cite this article as: Z. Luo, A.J. Rosenberg, H. Liu, J. Han, Z. Tu, Syntheses and in vitro
evaluation of new S1PR1 compounds and initial evaluation of a lead F-18 radiotracer in rodents,
European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.03.035.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical abstract
Syntheses and in vitro evaluation of new S1PR1 compounds and initial evaluation of
a lead F-18 radiotracer in rodents
Zonghua Luo, Adam J. Rosenberg, Hui Liu, Junbin Han, and Zhude Tu*
Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA
OH
N
N
N
O
N
N
18F
O
CF3
[18F]12b
= 9.7 ± 1.6 nM
S1PR1: IC50
> 1000 nM
S1PR2-5: IC50
LPS-treated mouse brain

ACCEPTED MANUSCRIPT
Syntheses and in vitro evaluation of new S1PR1 compounds and initial evaluation of
a lead F-18 radiotracer in rodents
Zonghua Luo, Adam J. Rosenberg, Hui Liu, Junbin Han, and Zhude Tu*
Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA
Total Pages: 33
Schemes: 5
Tables: 3
Figures: 5
*Address correspondence to:
Zhude Tu, Ph.D.
Department of Radiology
Washington University School of Medicine
Campus Box 8225
510 S. Kingshighway Blvd.
St. Louis, MO 63110, USA
Tel: 1-314-362-8487
Fax: 1-314-362-8555
E-mail: zhudetu@wustl.edu
Abstract
Thirteen new S1PR1 ligands were designed and synthesized by replacing azetidine-3-carboxylic acid moiety
of compound 4 with new polar groups. The in vitro binding potency of these new analogs toward S1PR1 was
1

ACCEPTED MANUSCRIPT
determined. Out of 13 new compounds, four compounds 9a, 10c, 12b, and 16b displayed high S1PR1 binding
potency with IC50 values of 13.2 ± 3.2, 14.7 ± 1.7, 9.7 ± 1.6, and 6.3 ± 1.3 nM, respectively; further binding
studies of these four ligands toward S1PR2-5 suggested they are highly selective for S1PR1 over other S1PRs.
The radiosynthesis of the lead radiotracer [18F]12b was achieved with good radiochemical yield (~14.1%), high
radiochemical purity (> 98%), and good specific activity (~54.1 GBq/µmol, decay corrected to the end of
synthesis, EOS). Ex vivo autoradiography and initial biodistribution studies in rodents were performed,
suggesting that [18F]12b was able to penetrate the blood-brain barrier (BBB) with high brain uptake (0.71%
ID/g at 60 min post injection) and no defluorination was observed. In vitro autoradiography study in brain slices
of lipopolysaccharides (LPS)-induced neuroinflammation mice indicated that SEW2871, a specific S1PR1
ligand was able to reduce the uptake of [18F]12b, suggesting [18F]12b has S1PR1 specific binding. These initial
results suggested that [18F]12b has potential to be a F-18 labeled radiotracer for imaging S1PR1 in the brain of
the animal in vivo.
Key words: Sphingosine-1-phosphate receptor 1; F-18 radiotracer; PET; Multiple sclerosis; Autoradiography;
Biodistribution.
1. Introduction
Sphingosine-1-phosphate receptor 1 (S1PR1) is a G-protein-coupled receptor which is widely expressed in
different cells such as immune cells, neural cells, endothelial cells, and smooth muscle cells. Acting with the
naturally occurring lipid mediator molecule sphingosine 1-phosphate (S1P, Fig. 1), S1PR1 initiates signaling
cascades that exert various biological functions, such as cell survival and differentiation, inflammation,
cardiovascular function, and vascular permeability [1]. It was reported that S1P/S1PR1 signaling system plays a
key role in immune-mediated diseases, such as multiple sclerosis (MS) [2, 3], colitis [4], influenza [5],
rheumatoid arthritis [6], inflammatory bowel disease [7], and atherosclerosis [8, 9]. In MS, S1PR1 involves in
immune-cell trafficking and activation, astrocyte proliferation, microglia activation, and interruption of the
BBB function [10, 11]. Fingolimod (FTY720, Fig. 1), an S1PR modulator, is the first oral drug that US Food
and Drug Administration (FDA) approved for treating remitting-relapsing MS [11]. In vivo, FTY720 is
2

ACCEPTED MANUSCRIPT
phosphorylated to form a bioactive metabolite, FTY720-P that exerts its disease-ameliorating effects by down-
modulation of S1PR1 expression from the cell surface and altering lymphocyte trafficking [10]. Currently,
targeting on S1PR1 represents a novel therapeutic strategy for MS and other inflammatory diseases.
Fig. 1. Structure of S1P, FTY720, and S1PR1 PET tracers.
Positron emission tomography (PET) provides a unique noninvasive imaging tool for quantitative
assessment of target protein in vivo and it is used widely in preclinical and clinical investigations of assessing
receptor occupancy, downstream functional changes, and diseases pathophysiology for guiding drug
development [12]. Using a specific S1PR1 radiotracer, PET could advance our understanding of the S1PR1
pathophysiologic functions in diseases. Utilizing a specific C-11 labeled S1PR1 radiotracer [11C]3 (Fig. 1), we
reported the feasibility of imaging measure S1PR1 expression for its inflammatory response in three animal
models of diseases, the experimental autoimmune encephalomyelitis (EAE) rat model of MS [13], the femoral
artery wire-injury mouse model of neointimal hyperplasia [14], and the carotid artery balloon injury rat model
of vascular inflammation [15]. Because F-18 isotope has a longer half-life (t1/2 = 109.8 min) than C-11 (t1/2
=
20.4 min), F-18 tracers provide additional advantages of fewer constraints for production, delivery to satellite
clinical PET sites with 3-4 hours’ drive distance, permission of longer scan sessions, and higher target-to-
reference ratios. Therefore, identification of a F-18 radiotracer for imaging S1PR1 in vivo is imperative in PET
community. To develop F-18 labeled S1PR1 radiotracers, Haufe’s group firstly reported three 18F-labeled FTY-
720 analogs as S1PR radiotracers [16, 17]. These F-18 labeled radiotracers were evaluated in rodents with PET
imaging modality for their inflammation responses by assessing S1PR1 expression at the binding sites.
However, in vivo defluorination and low selectivity of these radiotracers prevented further transferring these
3

ACCEPTED MANUSCRIPT
radiotracers into clinical evaluation. Recently, we reported our in vivo investigation of a selective F-18 S1PR1
tracer [18F]4 for its response to acute liver inflammation of rat model that was induced by injection of LPS [18].
Although it showed positive response in liver inflammation, [18F]4 is unable to penetrate the BBB, preventing
its implementation in the assessment of neuroinflammation in vivo. To overcome this limitation, herein, we
reported our efforts on design, syntheses, and in vitro bioactivity evaluation of 13 new analogs; among these 13
new analogs, the F-18 labeled [18F]12b was further radiosynthesized and performed initial evaluation to check
its specificity and capability of penetrating the BBB in rodents in vivo.
2. Results and discussion
2.1. Chemistry
Generally, the S1PR1 compound structure is composed of a lipophilic terminal group that interacts with the
hydrophobic binding pocket of S1PR1, a polar head group, and a rigid aryl or heteroaryl linker. Generally,
optimization of using a carboxylic acid as the polar head group lead to discovering high potent and highly
selective S1PR1 agonists [19]. The promising in vitro binding data of compound 4 that has an aryl azetidine
carboxylic acid group [18] encourages us to further optimize the aryl azetidine carboxylic acid group using other
hydrophilic groups to identify new analogs having improved binding properties and physicochemical properties.
In current work, we replaced the aryl azetidine carboxyl acid group with aryl sulfonamide, amide, N-hydroxy
amide, hydrazide, alcohol, carboxyl acid, and amine groups to generate new analogs. The syntheses of these
new analogs were illustrated in Scheme 1-4.
Compounds 9a-e were generated according to Scheme 1 by following reported procedure [18]. Alkylation
of commercially available 4-fluoro-3-(trifluoromethyl)benzoic acid 5 and 2-fluoroethan-1-ol generated the
intermediate 4-(2-fluoroethoxy)-3-(trifluoromethyl)benzoic acid 6 under the condition of sodium hydride in
dimethyl sulfoxide (DMSO). Meanwhile, the 4-position substituted amidoximes 8a-e were prepared from
corresponding benzonitriles 7a-e reacting with hydroxylamine hydrochloride and triethylamine. Coupling of 6
with 8a-e under the conventional peptide coupling conditions and subsequently thermally cyclization yielded
the oxadiazoles 9a-e.
4

ACCEPTED MANUSCRIPT
Scheme 1. Syntheses of 9a-e. Reagents and conditions: (a) NaH, 2-fluoroethan-1-ol, DMSO, 1 M HCl, RT; (b) hydroxylamine
hydrochloride, NaHCO3, methanol, reflux; (c) TBTU, HOBt, DIPEA, DMF, RT-120 °C.
The syntheses of compounds 10a-c were depicted in Scheme 2. Starting from compound 9d, compounds
10a-c were synthesized by the following procedures. The hydrolysis of 9d using lithium hydroxide as base gave
compound 10a, and the ammonolysis of 9d with hydroxylamine or hydrazine provided 10b or 10c, respectively.
Scheme 2. Syntheses of 10a-c. Reagents and conditions: (a) LiOH, THF, H2O, RT; (b)
hydroxylamine, KOH, methanol, reflux; (c) hydrazine hydrate, ethanol, reflux.
The syntheses of analogs 12a, 12b, 15, and 16a-b were illustrated in Scheme 3. The alcohol intermediate 9e
was easily transformed to azide 11a under the condition of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) and
diphenyl phosphoryl azide (DPPA), which was used to proceed the copper(II)-catalyzed alkyne cycloaddition to
afford compounds 12a-b. The alcohol intermediate 9e was converted to aldehyde 13 by Swern oxidation. After
reductive amination from aldehyde 13 using corresponding amines afforded compounds 14 and 16a-b.
Deprotection of tert-butyloxycarbonyl (Boc) group of 14 using 4 M HCl in dioxane afforded compound 15.
5

ACCEPTED MANUSCRIPT
Scheme 3. Syntheses of 12a-b, 15, and 16a-b. Reagents and conditions: (a) DPPA, DBU, toluene, 0 °C-RT; (b) oxalyl chloride, DMSO,
CH2Cl2, triethylamine, -78 °C-RT; (c) propiolic acid or propargyl alcohol, CuSO4•5H2O, sodium ascorbate, THF, H2O, RT; (d) NaBH3CN,
amines, acetic acid, methanol, RT; (e) 4-(N-Boc-amino)piperidine, NaBH(OAc)3, acetic acid, 1,2-dichloroethane , RT; (f) 4 M HCl in
dioxane, RT.
The syntheses of the analogs 21a-b were shown in Scheme 4. Amines 17a-b were protected using Boc
group to afford 18a-b. Oxadiazoles 20a-b were prepared via a similar procedure shown in Scheme 1, followed
by deprotection of Boc group yielded compounds 21a-b.
Scheme 4. Syntheses of 21a-b. Reagents and conditions: (a) di-tert-butyl dicarbonate, triethylamine, CH2Cl2, RT; (b)
hydroxylamine hydrochloride, NaHCO3, methanol, reflux; (c) TBTU, HOBt, DIPEA, 6, DMF, RT-120 °C; (d) 4 M HCl in
dioxane, RT.
2.2. In vitro binding assay
Newly synthesized compounds were firstly evaluated for binding potency toward S1PR1 by a [32P]S1P
competitive binding assay following published procedure [20], S1P was used as a reference compound. The
6

ACCEPTED MANUSCRIPT
binding potency was expressed as the IC50 value. As shown in Table 1, most of the new analogs have good
binding potency for S1PR1 with the IC50 values from 6.3 nM to 341 nM except compound 10a (> 1000 nM)
that has a carboxyl group. Compounds 9a and 9c, bearing sulfonamides group exhibited good binding potency
with the IC50 values of 13.2 and 40.0 nM, respectively. Compound 9b, having an amide group on the benzene
ring showed a reduced potency with an IC50 value of 67.1 nM compared to sulfonamide compounds 9a and 9c.
Introducing N-hydroxy amide or hydrazide group on the benzene ring displayed a different impact on S1PR1
binding potency. Compared to benzamide compound 9b, benzohydrazide compound 10c showed a 5-fold
decreased IC50 value of 14.7 nM, but N-hydroxybenzamide compound 10b exhibited a 4-fold increased IC50
value of 272 nM. Replacing the azetidine carboxylic acid with 1,2,3-triazole-4-carboxylic acid, 2-amino acetic
acid, and 3-amino propanoic acid resulted in different S1PR1 binding potency. Compared to the IC50 value of
compound 4 (2.6 nM [18]), 1,2,3-triazole-4-carboxylic acid containing compound 12a (341 nM) has 130-fold
increased IC50 value; the 2-amino acetic acid containing compound 16a (99.8 nM) has 38-fold increased IC50
value; 3-amino propanoic acid containing compound 16b (6.3 nM) has comparable binding potency. Compound
12b showed a 35-fold decreased IC50 value of 9.7 nM compared to the corresponding acid 12a (341 nM), which
is also comparable to compound 4. Replacing the azetidine carboxylic acid of compound 4 with different
amines were evaluated, and the piperidine-4-amine caused the most increased IC50 value, the IC50 value of
compound 15 was 156 nM; both methylamine and O-ethylamine caused increased IC50 values too (34.2 nM for
21a, 44.2 nM for 21b), but less increase than compound 15. The representative competitive binding curves of
compounds 12b, 16b, and S1P for S1PR1 were displayed in Fig. 2. Our in vitro binding data showed that
compounds 9a, 10c, 12b, and 16b were potent for S1PR1 with the IC50 values less than 20 nM. Consequentially,
their IC50 values of binding to other S1P receptor subtypes S1PR2, S1PR3, S1PR4, and S1PR5 were determined
to check the binding selectivity. The IC50 values of these four compounds binding toward S1PR1-5 were
displayed in table 2. Our data showed the IC50 values of compounds binding toward S1PR2-5 are > 1000 nM,
indicating all four compounds 9a, 10c, 12b, and 16b are selective for S1PR1 over S1PR2-5. The
physicochemical properties of compounds play crucial role in brain penetration, and studies showed marketed
CNS drugs were found to generally have smaller molecular weight (MW), higher calculated partition coefficient
7

ACCEPTED MANUSCRIPT
Table 1.
Structures and binding affinities (mean ± SD) of S1P and new compounds toward
S1PR1a,b.
S1PR1
Compd.
R
MW
ClogP
TPSA
HBD
IC50 (nM)
1.4 ± 0.3
379.5
431.4
395.3
4.1
3.4
3.7
113.0
103.3
86.3
4
1
1
S1P
9a
O
O
13.2 ± 3.2
S
NH2
67.1 ± 12.6
9b
40.0 ± 17.8
> 1000
459.4
396.3
411.3
3.7
4.9
3.3
89.4
80.5
92.5
1
1
2
9c
10a
10b
272 ± 65
14.7 ± 1.7
410.3
3.3
98.3
2
10c
341 ± 126
9.7 ± 1.6
156 ± 47
99.8 ± 12.2
6.3 ± 1.3
34.2 ± 7.1
44.2 ± 6.8
477.4
463.4
464.5
439.4
453.4
381.3
425.4
4.2
3.1
4.1
1.3
2.0
4.0
4.1
108.4
91.4
72.4
92.5
92.5
69.2
78.4
1
1
1
2
2
1
1
12a
12b
15
16a
16b
21a
21b
a IC50 values were determined at least 3 independent experiments, each run was performed
in duplicate; Assays for compounds which showed no activity (IC50 > 1000 nM) were only
b
repeated twice;
MW, ClogP, TPSA, and HBD were calculated by ChemDraw
Professional 16.0.1.4 (77) (PerkinElmer Informatics, Inc.).
8

ACCEPTED MANUSCRIPT
Fig. 2. The presentative competitive binding curves of compounds 12b,
16b, and S1P for S1PR1. A CHO cell membrane containing
recombinant human S1PR1 was used in a [32P]S1P competitive binding
assay to measure the binding affinities. S1P (fitted IC50 = 1.4 ± 0.3 nM)
12b (fitted IC50 = 9.7 ± 1.6 nM); and 16b (fitted IC50 = 6.3 ± 1.3 nM).
Table 2
The IC50 values (mean ± SD) of S1P, 9a, 10c, 12b, and 16b toward other S1PRsa
IC50 (nM)
Compd.
S1PR1
S1PR2
3.6 ± 0.5
> 1000
> 1000
> 1000
> 1000
S1PR3
0.4 ± 0.2
> 1000
> 1000
> 1000
> 1000
S1PR4
151 ± 82
> 1000
> 1000
> 1000
> 1000
S1PR5
3.1 ± 1.1
> 1000
> 1000
> 1000
> 1000
1.4 ± 0.3
13.2 ± 3.2
14.7 ± 1.7
9.7 ± 1.6
6.3 ± 1.3
S1P
9a
10c
12b
16b
aIC50 values were determined at least 3 independent experiments, each run was
performed in duplicate; Assays for compounds which showed no activity (IC50
1000 nM) were only repeated twice.
>
9

ACCEPTED MANUSCRIPT
(ClogP), fewer hydrogen bond donors (HBDs), and lower topological polar surface area (TPSA) compared with
non-CNS drugs [21-23]. A useful guideline was concluded as follows to assess the molecules for CNS drugs:
MW ≤ 470; 1 < ClogP ≤ 3; 40 < TPSA ≤ 90; and HBD ≤ 2 [24, 25]. Among these four compounds, both
compounds 12b and 16b have good MW (463.4 and 453.4, respectively), ClogP (3.1 and 2.0, respectively),
TPSA (91.4 and 92.5, respectively), and HBD (1 and 2, respectively) parameters for CNS drugs, suggesting the
F-18 labeled version counterparts may have the capability of penetrating the BBB and entering the brain.
Therefore, we further radiosynthesized [18F]12b and conducted preliminary in vivo and in vitro evaluation in
rats via autoradiography and ex vivo biodistribution study.
2.3. Radiosynthesis of [18F]12b
Compound 25, the precursor for radiolabeling [18F]12b, was prepared by following Scheme 5. The
cyclization of compound 8e with 4-hydroxy-3-(trifluoromethyl)benzoic acid yielded compound 22, which was
converted to azide 23 in the presence of DPPA and DBU. After alkylating with ethylene ditosylate, followed by
azide-alkyne cycloaddition, tosylate precursor 25 was obtained. Radiosynthesis of [18F]12b was achieved by
using the nucleophilic reaction between the tosylate precursor 25 and [18F]KF in acetonitrile with Kryptofix 222
followed by deprotection of methoxymethyl (MOM) group. After purification using semi-preparative high-
performance liquid chromatography (HPLC) combined with solid-phase extraction (SPE), [18F]12b was
formulated using 10% ethanol in 0.9% saline with high chemical and radiochemical purity (> 98%), good
radiochemical yields (14.1 ± 2.9%), and high specific activity (54.1 ± 8.3 GBq/µmol, n = 6, decay corrected to
EOS). The representative radio-UV analytical HPLC chromatograms (Fig. S1) and method were presented in
the supporting material. It took approximately 90 min for the total radiosynthesis.
10

ACCEPTED MANUSCRIPT
Scheme 5 Syntheses of the precursor 25 and [18F]12b. Reagents and conditions: (a) 4-hydroxy-3-(trifluoromethyl)benzoic acid, TBTU,
HOBt, DIPEA, DMF, RT-120 °C; (b) DPPA, DBU, toluene, 0 °C, RT; (c) ethylene ditosylate, K2CO3, CH3CN, 90 °C; (d) 3-
(methoxymethoxy)prop-1-yne, CuSO4•5H2O, sodium ascorbate, THF, H2O, RT; (e) [18F]KF, Kryptofix 222, K2CO3, CH3CN, 110 °C,
15 min, 6 M HCl, 110 °C, 5 min, 6 M NaOH.
2.4. Ex vivo autoradiography study of [18F]12b in normal rat
Ex vivo autoradiography study was performed to check if [18F]12b is able to cross the BBB in the rat. An
adult male Sprague-Dawley (SD) rat was tail intravenously (i.v.) injected with [18F]12b and euthanized at 60
min post-injection. The brain was immediately removed and snap-frozen followed by sectioning and mounting
on glass slides. The brain sections were exposed and the distribution of radioactivity was visualized by a Bio-
Imaging analyzer. As shown in Fig. 3, [18F]12b was able to cross the BBB and showed high uptake in the rat
brain, which is consistent to the expression of S1PR1 in the brain of rodents and human [26-28].
Fig. 3. Ex vivo autoradiography of [18F]12b in rat brain.
2.5. In vitro autoradiography study of [18F]12b in LPS-treated mice
To confirm the binding specificity of [18F]12b toward S1PR1, in vitro autoradiography studies were
performed using mouse brain slices that generated from LPS-induced murine neuroinflammation model, a
11

ACCEPTED MANUSCRIPT
widely used model for neuroinflammation, and the expression of S1PR1 was activated in the mouse brain
following the LPS injection according to the report [29]. SEW2871, an S1PR1-specific ligand (IC50 = 37 nM)
[30] was used as a blocking agent. Adult male C57BL/6 mice were euthanized at 24 h after receiving an
intraperitoneal injection of LPS, brain tissues were collected and sectioned. For the control study, the slices
were incubated with [18F]12b; for the blocking study, slices were incubated with [18F]12b in the presence of
SEW2871 at 10 µM of concentration. In vitro autoradiography study showed that SEW2871 was able to reduce
the uptake of [18F]12b by ~36%, suggesting the binding of [18F]12b toward S1PR1 is specific (Fig. 4).
Fig. 4. Representative in vitro autoradiographic images (A) and quantification of autoradiography
(B) of brain sections from LPS-induced neuroinflammation mice under control or blocking
condition. SEW2871, an S1PR1-specific ligand (IC50 = 37 nM), was used as the blocking agent.
2.6. Biodistribution study of [18F]12b in rats
To further investigate the tissue distribution of [18F]12b in rodents, the ex vivo biodistribution study was
performed in adult female Lewis rats. Animals were euthanized at 5, 30, 60, and 120 min post-injection. As
shown in the Table 3, the initial uptake (ID%/g) at 5 min post injection in blood, heart, lung, muscle, fat,
pancreas, spleen, kidney, liver, bone, thymus, and brain was 0.49, 1.65, 2.79, 0.39, 0.51, 1.96, 2.22, 3.43, 5.69,
0.55, 0.69, and 0.49, respectively. A rapid clearance of radioactivity was observed in tissues heart, lung, spleen,
kidney, and liver; uptake (ID%/g) was decreased to 1.19, 1.96, 1.36, 2.11, and 4.30 at 30 min from 1.65, 2.79,
2.22, 3.43, and 5.69 at 5 min, respectively. The radioactivity accumulation in the bone has no change from 5
min to 120 min, suggesting [13F]12b had no defluorination in vivo. A high uptake (ID%/g) of [18F]12b in the
12

ACCEPTED MANUSCRIPT
brain was observed compared to radiotracer [18F]4 [18]. The brain uptake (ID%/g) of [18F]12b was 0.49, 0.57,
0.71, and 0.61 at 5, 30, 60, and 120 min, respectively. In brain regions including cerebellum, brain stem, cortex,
striatum, thalamus, and hippocampus, [18F]12b had similar distribution, the uptake (ID%/g) in each region was
0.53, 0.50, 0.59, 0.47, 0.46, and 0.43, respectively at 5 min; the brain uptake at 60 min post injection reached
the maximum, then gradually washed out as shown in Fig. 5. Our data suggested that [18F]12b has the potential
to be a PET tracer for imaging S1PR1 expression in the brain in vivo.
Table 3.
Biodistribution of [18F]12b in female Lewis rats.(%ID/g values, mean ± SD, n = 4).
Tissue uptake (%ID/g)
Organs
5 min
30 min
60 min
120 min
Blood
Heart
0.49 ± 0.07
1.65 ± 0.27
2.79 ± 0.57
0.39 ± 0.04
0.51 ± 0.22
1.96 ± 0.56
2.22 ± 0.23
3.43 ± 0.31
5.69 ± 0.60
0.55 ± 0.05
0.69 ± 0.08
0.49 ± 0.04
0.42 ± 0.05
1.19 ± 0.06
1.96 ± 0.07
0.59 ± 0.05
0.62 ± 0.10
1.89 ± 0.16
1.36 ± 0.04
2.11 ± 0.08
4.30 ± 0.37
0.45 ± 0.03
0.83 ± 0.07
0.57 ± 0.02
0.36 ± 0.02
1.05 ± 0.08
1.67 ± 0.09
0.51 ± 0.02
0.69 ± 0.07
1.52 ± 0.25
1.27 ± 0.08
1.91 ± 0.10
4.21 ± 0.21
0.45 ± 0.01
0.82 ± 0.04
0.71 ± 0.03
0.35 ± 0.01
0.94 ± 0.06
1.54 ± 0.07
0.47 ± 0.02
0.57 ± 0.07
1.45 ± 0.15
1.14 ± 0.10
1.62 ± 0.09
3.66 ± 0.14
0.51 ± 0.05
0.78 ± 0.05
0.61 ± 0.05
Lung
Muscle
Fat
Pancreas
Spleen
Kidney
Liver
Bone
Thymus
Brain
13

ACCEPTED MANUSCRIPT
Fig. 5. Regional brain uptake of [18F]12b in female Lewis rats. Animals were sacrificed at 5, 30, 60, and
120 min post-injection. %ID/gram values (mean ± SD) with 4 rats per group.
3. Conclusion
In summary, we explored the structure of lead compound 4 to afford new S1PR1 compounds by replacing
the 3-N-azetedinyl carboxyl acid with different hydrophilic groups. Thirteen new analogs were synthesized and
their in vitro binding affinities were determined. Four compounds 9a, 10c, 12b, and 16b had high S1PR1
binding potency with IC50 values ranging from 6.3 to 15 nM and also displayed highly selective for S1PR1
versus other S1P receptor subtypes (IC50 > 1000 nM for S1PR2-5). Radiosynthesis of [18F]12b was
accomplished with good yield and high purity. Our initial evaluation of [18F]12b in rodent using
autoradiography and biodistribution studies indicated [18F]12b was able to penetrate the BBB and had good
brain retention; no defluorination was observed in vivo. In vitro autoradiography study in brain slices of LPS-
induced neuroinflammation mice indicated that SEW2871, a specific S1PR1 ligand was able to reduce the
uptake of [18F]12b, suggesting that [18F]12b had specific binding toward S1PR1. Further in vivo evaluation of
14

ACCEPTED MANUSCRIPT
[18F]12b in animal models of neuroinflammatory diseases warrants the suitability of [18F]12b to be an S1PR1
specific PET radiotracer for assessing inflammation response in CNS.
4. Experimental section
4.1. General
Commercially available starting materials, reagents, and solvents were used as purchased without further
purification unless otherwise stated. Reactions were monitored by thin-layer chromatography (TLC) carried out
on pre-coated glass plates of silica gel 60 F254 from EMD Chemicals Inc. Visualization was accomplished with
ultraviolet light (UV 254 nm). Compounds were purified by recrystallization or flash column chromatography
using 230–400 mesh silica gel purchased from Silicycle. Melting points were determined on a MEL-TEMP 3.0
1
13
apparatus without corrected. H NMR and C NMR spectra were received from Varian 400 MHz 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; Acetone-d6:
δ 2.05 ppm). High-resolution positive ion mass was acquired by a Bruker MaXis 4G Q-TOF mass spectrometer
with electrospray ionization source.
All animal experiments were conducted under Washington University’s Institutional Animal Care and Use
Committee (IACUC)-approved protocols in accordance with the US National Research Council’s Guide for the
Care and Use of Laboratory Animals.
4.2. Chemistry
4.2.1. Synthesis of 9a-e
4-(2-Fluoroethoxy)-3-(trifluoromethyl)benzoic acid (6). To a round-bottom flask equipped with a stir bar was
added 4-fluoro-3-(trifluoromethyl)benzoic acid (8.32 g, 40.0 mmol), 2-fluoroethanol (3.84 g, 60 mmol), and
DMSO (100 mL) under N2 atmosphere. NaH (2.07 g, 90.0 mmol) was added portion wise with stirring. The
reaction was stirred at room temperature overnight and monitored by TLC. The mixture was poured into ice-
15

ACCEPTED MANUSCRIPT
water (1 L) and acidified with 1 M HCl to give a precipitate. The resulting precipitate was filtered off, washed
1
with water and hexanes to give a tan solid (9.6 g, 95%). MP: 131-133 °C. H NMR (400 MHz, DMSO-d6) δ
13.18 (br, 1H, -COOH), 8.37 – 8.04 (m, 2H, -ArH), 7.45 – 7.32 (m, 1H, -ArH), 4.89 – 4.74 (m, 1H, -CH2F),
4.72 – 4.58 (m, 1H, -CH2F), 4.52 – 4.39 (m, 1H, -CH2O-), 4.38 – 4.29 (m, 1H, -CH2O-).
General procedure for the synthesis of 8a-e. To a round-bottom flask equipped with a stir bar was added 7a-e
(1.0 eq), hydroxylamine hydrochloride (2.0 eq), NaHCO3 (4.0 eq), and methanol (5.0 mL/mmol). The reaction
was refluxed and stirred in a pre-heated 75 °C oil-bath for 6 h. The reaction mixture was cooled to room
temperature, and the precipitate was filtered off and washed with methanol. The filtrate was concentrated in
vacuo without further purification.
(E)-N'-hydroxy-4-sulfamoylbenzimidamide (8a). Yield: 70%. MP: 217-219 °C. 1H NMR (400 MHz, DMSO-
d6) δ = 7.88-7.78 (m, 4H, -ArH), 5.93 (s, 2H, -SO2NH2).
1
(E)-4-(N'-hydroxycarbamimidoyl)benzamide (8b). Yield: 39%. MP: 221-224 °C. H NMR (400 MHz,
DMSO-d6) δ 9.78 (s, 1H, -OH), 7.98 (s, 1H,-NH2), 7.86 (d, J = 8.4 Hz, 2H, -ArH), 7.74 (d, J = 8.4 Hz, 2H, -
ArH), 7.38 (s, 1H, -NH2), 5.88 (s, 2H, -CONH2).
1
(E)-N'-hydroxy-4-(methylsulfonamidomethyl)benzimidamide (8c). Yield: 95%. MP: 178-180 °C. H NMR
(300 MHz, DMSO-d6) δ 9.62 (s, 1H, -OH), 7.63 (d, J = 8.3 Hz, 2H, -ArH), 7.32 (d, J = 8.4 Hz, 2H, -ArH), 5.78
(s, 2H, -NH2), 4.14 (s, 2H, -CH2-), 2.84 (s, 3H, -CH3).
Methyl (E)-4-(N'-hydroxycarbamimidoyl)benzoate (8d). Yield: 86%. MP: 168-170 °C. 1H NMR (400 MHz,
DMSO-d6) δ = 9.91 (s, 1H, -OH), 7.94 (d, J = 8.0 Hz, 2H, -ArH), 7.82 (d, J = 8.4 Hz, 2H, -ArH), 5.93 (s, 2H, -
NH2), 3.85 (s, 3H, -OCH3)
1
(E)-N'-hydroxy-4-(hydroxymethyl)benzimidamide (8e). Yield: 76%. MP: 152-155 °C. H NMR (400 MHz,
DMSO-d6) δ 9.57 (s, 1H, -OH), 7.71 – 7.57 (m, 2H, -ArH), 7.37 – 7.24 (m, 2H, -ArH), 5.77 (s, 2H, -NH2), 5.24
(s, 1H, -CH2OH), 4.51 (s, 2H, -CH2-).
General procedure for the synthesis of 9a-e. To a round-bottom flask equipped with a stir bar was added acid
6 (1.0 eq), HOBt (0.2 eq), TBTU (1.0 eq), DIPEA (3.0 eq), and DMF (5.0 mL/mmol). The reaction mixture was
stirred for 0.5 h followed by adding amidoxime 8a-e (1.0 eq). The reaction mixture was stirred for 1 h at room
16

ACCEPTED MANUSCRIPT
temperature, then refluxed in a pre-heated 120 °C oil-bath for 4 h and monitored by TLC. After cooling, the
reaction mixture was diluted with water and extracted with ethyl acetate. The ethyl acetate layer was washed
with 1 M HCl, saturated brine, and dried over anhydrous MgSO4. After filtration and concentration, the residue
was purified on a silica gel column.
4-(5-(4-(2-Fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzenesulfonamide (9a). Yield:
51%. MP: 198-199 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.45 – 8.38 (m, 1H, -ArH), 8.33 – 8.24 (m, 3H, -ArH),
8.04 (d, J = 8.4 Hz, 2H, -ArH), 7.62 – 7.52 (m, 3H, -ArH&-NH2), 4.89 – 4.81 (m, 1H, -CH2F), 4.76 – 4.70 (m,
13
1H, -CH2F), 4.61 – 4.55 (m, 1H, -CH2O-), 4.54 – 4.44 (m, 1H, -CH2O-). C NMR (101 MHz, DMSO-d6) δ
174.92, 167.79, 160.02, 147.12, 134.58, 129.34, 128.21, 127.08 (q, J = 5.1 Hz), 127.04, 123.31 (d, J = 272.7
Hz), 118.61 (q, J = 31.3 Hz), 116.10, 115.44, 82.10 (d, J = 168.7 Hz), 69.24 (d, J = 18.2 Hz). HRMS (ESI)
calcd for C17H13F4N3O4S [M + H+] 432.0636. Found [M + H+] 432.0640.
4-(5-(4-(2-Fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzamide (9b). Yield: 29%. MP:
244-245 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.40 (d, J = 8.1 Hz, 1H, -ArH), 8.30 (s, 1H, -ArH), 8.19 – 8.10
(m, 3H, -ArH&-NH2), 8.10 – 8.03 (m, 2H, -ArH), 7.59 – 7.51 (m, 2H, -ArH), 4.89 – 4.82 (m, 1H, -CH2F), 4.76
13
– 4.71 (m, 1H, -CH2F), 4.62 – 4.55 (m, 1H, -CH2O-), 4.53 – 4.47 (m, 1H, -CH2O-). C NMR (101 MHz,
DMSO-d6) δ 174.72, 168.14, 167.49, 159.96, 137.42, 134.54, 128.77, 127.42, 127.08 (q, J = 5.1 Hz), 123.31 (d,
J = 273.7 Hz), 118.58 (q, J = 31.3 Hz), 116.17, 115.41, 82.10 (d, J = 168.7 Hz), 69.23 (d, J = 19.2 Hz). HRMS
(ESI) calcd for C18H13F4N3O3 [M + H+] 396. 0966. Found [M + H+] 396.0959.
N-(4-(5-(4-(2-Fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzyl)methanesulfonamide
(9c). Yield: 9%. MP: 208-300 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.41 (d, J = 8.9 Hz, 1H, -ArH), 8.31 (s, 1H,
-ArH), 8.07 (d, J = 8.1 Hz, 2H, -ArH), 7.73 – 7.67 (m, 1H, -ArH), 7.61 – 7.52 (m, 3H, -ArH&-NHSO2-), 4.89 –
4.81 (m, 1H, -CH2F), 4.78 – 4.70 (m, 1H, -CH2F), 4.63 – 4.56 (m, 1H, -CH2O-), 4.55 – 4.48 (m, 1H, -CH2O-),
13
4.26 (d, J = 6.3 Hz, 2H, -ArCH2-), 2.91 (s, 3H, -SO2CH3). C NMR (101 MHz, DMSO-d6) δ 174.54, 168.46,
159.91, 142.71, 134.54, 128.77, 127.64, 127.07 (q, J = 5.1 Hz), 125.29, 123.34 (d, J = 273.7 Hz), 118.56 (q, J =
31.3 Hz), 116.31, 115.43, 82.12 (d, J = 168.7 Hz), 69.23 (d, J = 18.2 Hz), 46.13, 40.35. HRMS (ESI) calcd for
C19H17F4N3O4S [M + H+] 460.0949. Found [M + H+] 460.0936.
17

ACCEPTED MANUSCRIPT
Methyl 4-(5-(4-(2-fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzoate (9d). Yield: 62%.
MP: 166-169 °C. 1H NMR (400 MHz, Acetone-d6) δ 8.45 (d, J = 8.9 Hz, 1H, -ArH), 8.42 (s, 1H, -ArH), 8.26 (d,
J = 8.1 Hz, 2H, -ArH), 8.18 (d, J = 8.2 Hz, 2H, -ArH), 7.57 (d, J = 8.7 Hz, 1H, -ArH), 4.96 – 4.92 (m, 1H, -
CH2F), 4.85 – 4.80 (m, 1H, -CH2F), 4.68 – 4.64 (m, 1H, -CH2O-), 4.62 – 4.56 (m, 1H, -CH2O-), 3.94 (s, 3H, -
OCH3). 13C NMR (101 MHz, Acetone-d6) δ 174.80, 168.03, 165.62, 160.06, 133.86, 132.69, 130.76, 129.94,
127.34, 126.97 (q, J = 5.1 Hz), 123.12 (d, J = 273.7 Hz), 119.22 (d, J = 31.3 Hz), 116.41, 114.50, 81.54 (d, J =
169.7 Hz), 68.95 (d, J = 19.2 Hz), 51.75.
(4-(5-(4-(2-Fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)phenyl)methanol (9e) [18].
Yield: 65%. MP: 190-192 °C. 1H NMR (400 MHz, CDCl3) δ = 8.45 (d, J = 2.0 Hz, 1H, -ArH), 8.33 (dd, J = 8.8
Hz, 2.0 Hz, 1H, -ArH), 8.13 (d, J = 8.0 Hz, 2H, -ArH), 7.50 (d, J = 8.0 Hz, 2H, -ArH), 7.16 (d, J = 8.4 Hz, 1H, -
ArH), 4.90−4.74 (m, 4H, -CH2F &-CH2OH), 4.48−4.35 (m, 2H, -CH2O-).
4.2.2. Synthesis of 10a-c
4-(5-(4-(2-Fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzoic acid (10a). To a round-
bottom flask equipped with a stir bar was added ester 9d (2.0 g, 4.87 mmol), lithium hydroxide (230 mg, 9.75
mmol), THF (20 mL), and water (4 mL). The reaction mixture was stirred overnight and acidified with 1 M HCl
to pH = 1. The reaction mixture was extracted with ethyl acetate (20 mL), and the ethyl acetate layer was
washed with saturated brine, and dried over anhydrous MgSO4. After filtration and concentration, 10a was
obtained as an off-white solid. (1.7 g, 87%). MP: 220-222 °C. 1H NMR (400 MHz, DMSO-d6) δ 13.26 (s, 1H, -
COOH), 8.39 (d, J = 8.8 Hz, 1H, -ArH), 8.28 (s, 1H, -ArH), 8.19 – 8.08 (m, 4H, -ArH), 7.54 (d, J = 8.8 Hz, 1H,
-ArH), 4.91 – 4.81 (m, 1H, -CH2F), 4.79 – 4.69 (m, 1H, -CH2F), 4.63 – 4.47 (m, 2H, -CH2O-). 13C NMR (101
MHz, DMSO-d6) δ 174.80, 168.02, 167.02, 159.98, 134.55, 133.84, 130.13, 129.85, 127.70, 127.07 (q, J = 5.1
Hz), 123.30 (d, J = 273.7 Hz), 118.57 (q, J = 31.3 Hz), 116.11, 115.41, 82.09 (d, J = 168.7 Hz), 69.22 (d, J =
19.2 Hz).
4-(5-(4-(2-Fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)-N-hydroxybenzamide (10b). To
a round-bottom flask equipped with a stir bar was added ester 9d (150 mg, 0.37 mmol), hydroxylamine
18

ACCEPTED MANUSCRIPT
hydrochloride (76 mg, 1.1 mmol) and methanol (1.9 mL), followed by adding potassium hydroxide (12.5 mg,
2.22 mmol). The reaction mixture was refluxed overnight. After cooling to room temperature, the mixture was
diluted with water (20 mL) to precipitate the product. The precipitate was filtered off and washed with diethyl
1
ether to give an off-white solid (111 mg, 73%). MP: 186-189 °C. H NMR (400 MHz, DMSO-d6) δ 11.43 (s,
1H, -OH), 9.20 (s, 1H, -NH-), 8.42 (d, J = 8.1 Hz, 1H, -ArH), 8.31 (s, 1H, -ArH), 8.18 – 8.11 (m, 2H, -ArH),
8.01 – 7.91 (m, 2H, -ArH), 7.61 – 7.51 (m, 1H, -ArH), 4.92 – 4.68 (m, 2H, -CH2F), 4.64 – 4.47 (m, 2H, -CH2O-
13
). C NMR (101 MHz, DMSO-d6) δ 174.77, 168.12, 167.05, 159.99, 135.95, 134.59, 130.55, 128.23, 127.59,
127.12 (d, J = 5.1 Hz), 123.31 (d, J = 274.7 Hz), 118.57 (J = 30.3 Hz), 116.19, 115.45, 82.11 (d, J = 168.7 Hz),
69.24 (d, J = 19.2 Hz). HRMS (ESI) calcd for C18H13F4N3O4 [M + H+] 412.0919. Found [M + H+] 412.0915.
4-(5-(4-(2-Fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzohydrazide (10c). To a
round-bottom flask equipped with a stir bar was added ester 9d (205 mg, 0.5 mmol), ethanol (1.3 mL), and
hydrazine monohydrate (0.07 mL, 1.5 mmol). The reaction mixture was refluxed overnight. After cooling to
room temperature, the precipitate was filtered off and washed with diethyl ether to give an off-white solid. (100
mg, 49%). MP: 186-188 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.98 (s, 1H, -CONH-), 8.42 (d, J = 8.7 Hz, 1H, -
ArH), 8.32 (s, 1H, -ArH), 8.14 (d, J = 8.2 Hz, 2H, -ArH), 8.01 (d, J = 8.2 Hz, 2H, -ArH), 7.56 (d, J = 8.8 Hz, 1H,
-ArH), 4.89 – 4.81 (m, 1H, -CH2F), 4.77 – 4.70 (m, 1H, -CH2F), 4.68 – 4.55 (m, 3H, -CH2O-&-NH2), 4.55 –
4.48 (m, 1H, -CH2O-). 13C NMR (101 MHz, DMSO-d6) δ 174.75, 168.14, 165.35, 159.98, 136.46, 134.57,
128.62, 128.30, 127.52, 127.11 (q, J = 5.1 Hz), 123.32 (d, J = 273.7 Hz), 118.60 (q, J = 31.3 Hz), 116.20,
115.46, 82.11 (d, J = 167.7 Hz), 69.25 (d, J = 19.2 Hz). HRMS (ESI) calcd for C18H14F4N4O3 [M + H+] 411.
1075. Found [M + H+] 411.1067.
4.2.3. Synthesis of 12a-b, 15, and 16a-b
3-(4-(Azidomethyl)phenyl)-5-(4-(2-fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazole (11a). To a
round-bottom flask equipped with a stir bar was added 9e (1.0 g, 2.62 mmol), diphenyl phosphoryl azide (0.86 g,
3.14 mmol), and toluene (5.0 mL). The mixture was cooled to 0 °C before adding 1,8-diazabicyclo[5.4.0]undec-
7-ene (0.48, 3.14 mmol) dropwise. The reaction was warmed to room temperature slowly and stirred overnight.
19

ACCEPTED MANUSCRIPT
Then, the reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (30 mL). The ethyl
acetate layer was washed with 1 M HCl, saturated brine and dried over anhydrous MgSO4. After filtration and
concentration, the residue was purified on a silica gel column, eluted with ethyl acetate/hexanes (V/V, 3/7) to
give a white solid. (330 mg, 62%). MP: 117-119 °C. 1H NMR (400 MHz, CDCl3) δ = 8.47 (d, J = 2.0 Hz, 1H, -
ArH), 8.35 (dd, J = 8.8 Hz, 2.0 Hz, 1H, -ArH), 8.19 (d, J = 8.4 Hz, 2H, -ArH), 7.47 (d, J = 8.0 Hz, 2H, -ArH),
7.18 (d, J = 8.8 Hz, 1H, -ArH), 4.94 – 4.73 (m, 2H, -CH2F), 4.50 – 4.35 (m, 4H, -CH2O-&-CH2N3). 13C NMR
(101 MHz, Acetone-d6) δ 174.54, 168.40, 159.98, 139.55, 133.81, 129.35, 128.89, 127.60, 126.92 (q, J = 5.1
Hz), 123.15 (d, J = 273.7 Hz), 119.21 (d, J = 31.3 Hz), 116.60, 114.50, 81.55 (d, J = 169.7 Hz), 68.94 (d, J =
20.2 Hz), 53.69.
General procedure for the synthesis of 12a-b. To a round-bottom flask equipped with a stir bar was added
azide 11a (1.0 eq) and THF. Once the azide was dissolved, water (0.6 mL/mmol) was added followed by
propiolic acid or propargyl alcohol (1.05 eq), sodium ascorbate (0.10 eq), and copper sulfate pentahydrate (0.06
eq). The reaction mixture was stirred for 4 h and monitored by TLC. Then, the reaction mixture was diluted
with water and extracted with ethyl acetate. The ethyl acetate layer was washed with saturated brine and dried
over anhydrous MgSO4. After filtration and concentration, a yellow solid was obtained.
1-(4-(5-(4-(2-Fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzyl)-1H-1,2,3-triazole-4-
carbox-ylic acid (12a). MP: 157-158 °C. 1H NMR (400 MHz, DMSO-d6) δ 13.09 (s, 1H, -COOH), 8.86 (s, 1H,
-ArH), 8.41 (d, J = 8.2 Hz, 1H, -ArH), 8.30 (s, 1H, -ArH), 8.10 (d, J = 7.5 Hz, 2H, -ArH), 7.53 (d, J = 7.7 Hz,
2H, -ArH), 7.17 – 7.11 (m, 1H, -ArH), 5.78 (s, 2H, -ArCH2-), 4.88 – 4.46 (m, 4H, -OCH2CH2F). 13C NMR (101
MHz, DMSO-d6) δ 174.64, 168.26, 162.01, 159.93, 140.38, 139.55, 134.57, 129.31, 129.20, 128.04, 127.08 (d,
J = 5.1 Hz), 123.32 (d, J = 274.7 Hz), 120.33, 118.56 (q, J = 31.3 Hz), 116.24, 115.43, 82.11 (d, J = 167.7 Hz),
69.23 (d, J = 19.2 Hz), 53.04. HRMS (ESI) calcd for C21H15F4N5O4 [M + H+] 478.1133. Found [M + H+]
478.1125.
(1-(4-(5-(4-(2-Fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzyl)-1H-1,2,3-triazol-4-
yl)met-hanol (12b). MP: 175-177 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.41 (d, J = 8.6 Hz, 1H, -ArH), 8.30 (s,
1H, -ArH), 8.08 (d, J = 4.7 Hz, 3H, -ArH), 7.60 – 7.45 (m, 3H, -ArH), 5.70 (s, 2H, -ArCH2-), 5.20 (s, 1H, -OH),
20

ACCEPTED MANUSCRIPT
4.89 – 4.67 (m, 2H, -CH2F), 4.63 – 4.48 (m, 4H, -CH2O-&-CH2OH). 13C NMR (101 MHz, DMSO-d6) δ 174.63,
168.29, 159.94, 140.24, 134.57, 129.11, 127.97, 127.08 (d, J = 5.1 Hz), 126.13, 123.32 (d, J = 274.7 Hz),
123.55, 120.31, 118.56 (q, J = 31.3 Hz), 116.24, 115.43, 82.11 (d, J = 167.7 Hz), 69.23 (d, J = 19.2 Hz), 55.47,
52.71. HRMS (ESI) calcd for C21H17F4N5O3 [M + H+] 464.1346. Found [M + H+] 464.1353.
4-(5-(4-(2-Fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzaldehyde (13) [18]. To an
oven-dried 100 mL round-bottom flask equipped with a stir bar was added CH2Cl2 (32 mL) and DMSO (1.7 g,
21.7 mmol). The reaction mixture was cooled to -78 °C and oxalyl chloride (1.87 g, 14.7 mmol) was added
slowly under N2 atmosphere. The reaction mixture was stirred for 30 minutes at which time 9e (2.68 g, 7.0
mmol) was added. The reaction mixture was stirred for another 30 minutes at which time triethylamine (5.67
mL, 56.0 mmol) was added. The cooling bath was removed and the reaction was allowed to warm to room
temperature over 2 h. The reaction mixture was concentrated in vacuo and partitioned between ethyl acetate and
1 M HCl. The layers were separated and the aqueous layer was re-extracted with ethyl acetate. The combined
organic layers were washed with 1 M HCl, sat. NaHCO3, brine, and dried over MgSO4. After filtration and
concentration in vacuo, a pale-yellow solid was obtained. (1.51 g, 76%).
tert-Butyl (1-(4-(5-(4-(2-fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzyl)piperidin-4-
yl)carba-mate (14). To a round-bottom flask equipped with a stir bar was added aldehyde 13 (380 mg, 1.0
mmol), amine (21 mg, 1.05 mmol), 1,2-dichloroethane (10 mL), sodium triacetoxyborohydride (318 mg, 1.5
mmol), and acetic acid (120 mg, 2.0 mmol). The reaction mixture was stirred overnight at room temperature.
Then, the reaction was quenched with saturated NaHCO3 aqueous and extracted with ethyl acetate (20 mL × 3).
The combined ethyl acetate layer was washed with brine and dried over anhydrous MgSO4. After filtration and
concentration, the crude product was purified on a silica gel column to give white solid product (250 mg, 44%).
MP: 147-149 °C. 1H NMR (400 MHz, Acetone-d6) δ 8.39 – 8.32 (m, 2H, -ArH), 8.02 (d, J = 7.9 Hz, 2H, -ArH),
7.52 – 7.45 (m, 3H, -ArH), 5.83 (d, J = 6.7 Hz, 1H, -ArH), 4.90 – 4.83 (m, 1H, -CH2F), 4.79 – 4.72 (m, 1H, -
CH2F), 4.61 – 4.56 (m, 1H, -CH2O-), 4.55 – 4.49 (m, 1H, -CH2O-), 3.51 (s, 2H, -ArCH2-), 3.41 – 3.27 (m, 1H, -
CHNH-), 2.83 – 2.75 (m, 2H, -NCH2-), 2.12 – 1.97 (m, 2H, -NCH2-), 1.83 – 1.75 (m, 2H, -CH2-), 1.55 – 1.43
(m, 2H, -CH2-), 1.34 (s, 9H, -CH3). 13C NMR (101 MHz, Acetone-d6) δ 174.29, 168.62, 159.90, 159.89, 155.00,
21

ACCEPTED MANUSCRIPT
143.07, 133.74, 129.26, 127.11, 126.86 (q, J = 5.1 Hz), 125.33, 123.15 (d, J = 273.7 Hz), 119.17 (q, J = 31.3
Hz), 116.65, 114.43, 81.54 (d, J = 169.7 Hz), 77.51, 68.91 (d, J = 20.2 Hz), 62.18, 52.44, 47.89, 32.27, 27.75.
1-(4-(5-(4-(2-Fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzyl)piperidin-4-amine
dihydro-chloride (15). To a round-bottom flask equipped with a stir bar was added the Boc-protected amine 14
(90 mg, 0.16 mmol) and 2 mL of 4 M HCl in 1,4-dioxane. The reaction was stirred 4 h at room temperature, and
the precipitate was filtered off, washed with ethyl ether to give a white solid. (51 mg, 59%). MP: 268-269 °C.
1H NMR (400 MHz, DMSO-d6) δ 8.50 (s, 2H, -NH2), 8.42 (d, J = 9.0 Hz, 1H, -ArH), 8.31 (s, 1H, -ArH), 8.14
(d, J = 7.9 Hz, 2H, -ArH), 7.85 (d, J = 7.4 Hz, 2H, -ArH), 7.58 (d, J = 8.8 Hz, 1H, -ArH), 4.90 – 4.81 (m, 1H, -
CH2F), 4.78 – 4.70 (m, 1H, -CH2F), 4.63 – 4.56 (m, 1H, -CH2O-), 4.55 – 4.49 (m, 1H, -CH2O-), 4.36 (s, 2H, -
ArCH2-), 3.36 – 3.17 (m, 3H, -N-CH2-&-CHNH2), 3.16 – 3.00 (m, 2H, -N-CH2-), 2.22 – 1.95 (m, 4H, -CH2CH-).
13C NMR (101 MHz, DMSO-d6) δ 174.75, 168.26, 159.99, 134.60, 133.60, 132.92, 127.81, 127.36, 127.10 (d,
J = 5.1 Hz), 123.32 (d, J = 273.7 Hz), 118.58 (q, J = 31.3 Hz), 116.20, 115.52, 82.13 (d, J = 168.7 Hz), 69.27 (d,
J = 18.2 Hz), 58.66, 49.97, 45.68, 27.21.HRMS (ESI) calcd for C23H24F4N4O2 [M + H+] 465.1908. Found [M +
H+] 465.1907.
General procedure for the synthesis of 16a-b. To a round-bottom flask equipped with a stir bar was added
aldehyde 13 (1.0 eq), amine (1.5 eq), methanol (14 mL/mmol), and acetic acid (0.5 mL/mmol). The reaction
mixture was stirred 1 h at which time sodium cyanoborohydride (1.0 eq) was added. The reaction mixture was
stirred overnight and diluted with water. The precipitate was filtered off and washed with water to give an off-
white solid product.
(4-(5-(4-(2-Fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzyl)glycine (16a). Yield: 24%.
MP: 175-176 °C. 1H NMR (400 MHz, Acetone-d6) δ 8.49 – 8.35 (m, 2H, -ArH), 8.12 (d, J = 7.8 Hz, 2H, -ArH),
7.67 (d, J = 7.9 Hz, 2H, -ArH), 7.56 (d, J = 8.7 Hz, 1H, -ArH), 4.98 – 4.75 (m, 2H, -CH2F), 4.71 – 4.54 (m, 2H,
-CH2O-), 3.98 (s, 2H, -ArCH2-), 3.42 (s, 2H, -CH2COOH). 13C NMR (101 MHz, Acetone-d6) δ 174.36, 171.35,
168.62, 159.93, 143.12, 133.77, 129.47, 127.27, 126.88 (q, J = 5.1 Hz), 125.65, 123.15 (d, J = 273.7 Hz),
119.19 (q, J = 31.3 Hz), 116.66, 114.47, 81.56 (d, J = 168.7 Hz), 68.92 (d, J = 20.2 Hz), 57.26, 53.12. HRMS
(ESI) calcd for C20H17F4N3O4 [M + H+] 440.1228. Found [M + H+] 440.1210.
22

ACCEPTED MANUSCRIPT
3-((4-(5-(4-(2-Fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzyl)amino)propanoic
acid (16b). Yield: 17%. MP: 198-200 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.49 – 8.37 (m, 1H, -ArH), 8.30 (s,
1H, -ArH), 8.17 – 7.98 (m, 2H, -ArH), 7.67 – 7.44 (m, 3H, -ArH), 4.92 – 4.70 (m, 2H, -CH2F), 4.63 – 4.45 (m,
13
2H, -CH2O-), 3.87 (s, 2H, -ArCH2-), 2.87 – 2.71 (m, 2H, -NHCH2-), 2.45 – 2.31 (m, 2H, -CH2COOH). C
NMR (101 MHz, DMSO-d6) δ 174.48, 174.15, 168.53, 159.91, 143.50, 134.53, 129.37, 127.52, 127.05 (d, J =
5.1 Hz), 125.10, 123.33 (d, J = 5.1 Hz), 118.56 (d, J = 273.7 Hz), 116.31, 115.43, 82.12 (d, J = 168.7 Hz),
69.24 (d, J = 19.2 Hz), 51.89, 44.51, 34.00. HRMS (ESI) calcd for C21H19F4N3O4 [M + H+] 454.1384. Found
[M + H+] 454.1370.
4.2.4. Synthesis of 21a-b
General procedure for the synthesis of 18a-b. To a round-bottom flask equipped with a stir bar was added
17a-b (1.0 eq), Boc2O (1.1 eq), CH2Cl2 (10 mL/mmol), and triethylamine (1.5 eq). The reaction mixture was
stirred at room temperature overnight. The reaction was concentrated in vacuo and partitioned between ethyl
ether and 1 M HCl. The organic layer were separated, washed with saturated NaHCO3, brine, dried over MgSO4,
and concentrated in vacuo to give a white crystalline solid.
tert-Butyl (4-cyanobenzyl)carbamate (18a). Yield: 93%. MP: 114-116 °C. 1H NMR (400 MHz, CDCl3) δ 7.62
(d, J = 7.9 Hz, 2H, -ArH), 7.39 (d, J = 8.5 Hz, 2H, -ArH), 4.96 (s, 1H, -NH-), 4.37 (d, J = 5.9 Hz, 2H, -CH2-),
1.46 (s, 9H, -CH3).
tert-Butyl (2-((4-cyanobenzyl)oxy)ethyl)carbamate (18b). Yield: 83%. MP: 100-102 °C. 1H NMR (400 MHz,
CDCl3) δ 7.62 (d, J = 7.9 Hz, 2H, -ArH), 7.33 (d, J = 7.9 Hz, 2H, -ArH), 4.53 (s, 2H, -ArCH2-), 3.80 – 3.65 (m,
2H, -OCH2-), 3.49 – 3.30 (m, 2H, -CH2NH-), 1.40 (s, 9H, -CH3).
General procedure for the synthesis of 19a-b. To a round-bottom flask equipped with a stir bar was added
18a-b (1.0 eq), hydroxylamine hydrochloride (2.0 eq), NaHCO3 (4.0 eq), and methanol (10 mL/mmol). The
reaction was refluxed and stirred in a pre-heated 75 °C oil-bath for 6 h. After cooling to room temperature, and
the precipitate was filtered off and washed with methanol. The filtrate was concentrated in vacuo and further
purified on a silica gel column.
23

ACCEPTED MANUSCRIPT
1
tert-Butyl (E)-(4-(N'-hydroxycarbamimidoyl)benzyl)carbamate (19a). Yield: 65%. MP: 142-145 °C. H
NMR (400 MHz, DMSO-d6) δ 9.57 (s, 1H, -OH), 7.61 (d, J = 8.1 Hz, 2H, -ArH), 7.40 (t, J = 6.1 Hz, 1H, -
NHCO-), 7.22 (d, J = 8.1 Hz, 2H, -ArH), 5.77 (s, 2H, -NH2), 4.13 (d, J = 6.1 Hz, 2H, -ArCH2-), 1.40 (s, 9H, -
CH3).
tert-Butyl (E)-(2-((4-(N'-hydroxycarbamimidoyl)benzyl)oxy)ethyl)carbamate (19b). Yield: 40%. MP: 138-
140 °C. 1H NMR (400 MHz, Acetone-d6) δ 8.87 (s, 1H, -OH), 7.69 (d, J = 7.7 Hz, 2H, -ArH), 7.28 (d, J = 8.1
Hz, 2H, -ArH), 5.45 (s, 2H, -NH2), 4.54 (s, 2H, -ArCH2-), 3.75 (s, 1H, -NHCO-), 3.68 – 3.59 (m, 2H, -OCH2-),
3.40 – 3.24 (m, 2H, -CH2NH-), 1.47 (s, 9H, -CH3).
General procedure for the synthesis of 20a-b. To a round-bottom flask equipped with a stir bar was added
acid 6 (1.0 eq), HOBt (0.2 eq), TBTU (1.0 eq), DMF (5 mL/mmol), and DIPEA (3.0 eq). The reaction mixture
was stirred for 0.5 h before adding amidoxime 19a-b (1.0 eq). The reaction mixture was stirred for 1 h at room
temperature, then heated in a pre-heated 120 °C oil-bath. The reaction mixture was stirred for 4 h and monitored
by TLC. After cooling, the reaction mixture was diluted with water and extracted with ethyl acetate. The ethyl
acetate layer was washed with 1 M HCl, saturated brine, and dried over anhydrous MgSO4. After filtration and
concentration, the residue was purified on a silica gel column.
tert-Butyl
(4-(5-(4-(2-fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzyl)carbamate
(20a). Yield: 56%. MP: 158-160 °C. 1H NMR (400 MHz, DMSO-d6) δ 8.40 (d, J = 9.0 Hz, 1H, -ArH), 8.30 (s,
1H, -ArH), 8.03 (d, J = 8.0 Hz, 2H, -ArH), 7.58 – 7.47 (m, 2H, -ArH&-NHCO), 7.44 (d, J = 8.0 Hz, 2H, -ArH),
4.88 – 4.82 (m, 1H, -CH2F), 4.76 – 4.70 (m, 1H, -CH2F), 4.61 – 4.55 (m, 1H, -CH2O-), 4.54 – 4.48 (m, 1H, -
CH2O-), 4.22 (d, J = 5.9 Hz, 2H, -ArCH2-), 1.41 (s, 9H, -CH3). 13C NMR (101 MHz, DMSO-d6) δ 174.45,
168.52, 159.88, 156.27, 144.53, 134.49, 128.04, 127.54, 127.04 (q, J = 5.1 Hz), 123.33 (d, J = 273.7 Hz),
121.97, 118.56 (d, J = 30.3 Hz), 116.32, 115.40, 82.11 (d, J = 168.7 Hz), 78.39, 69.22 (d, J = 19.2 Hz), 43.66,
28.65.
tert-Butyl (2-((4-(5-(4-(2-fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzyl)oxy)ethyl)-
carbamate (20b). Yield: 42%. MP: 144-147 °C. 1H NMR (400 MHz, CDCl3) δ 8.47 (s, 1H, -ArH), 8.35 (d, J =
8.7 Hz, 1H, -ArH), 8.13 (d, J = 8.1 Hz, 2H, -ArH), 7.38 (d, J = 7.8 Hz, 2H, -ArH), 7.18 (d, J = 8.8 Hz, 1H, -
24

ACCEPTED MANUSCRIPT
ArH), 4.92 – 4.86 (m, 1H, -CH2F), 4.80 – 4.75 (m, 1H, -CH2F), 4.56 (s, 2H, -ArCH2-), 4.49 – 4.44 (m, 1H, -
CH2O-), 4.42 – 4.37 (m, 1H, -CH2OAr-), 3.80 – 3.69 (m, 2H, -OCH2-), 3.52 – 3.35 (m, 2H, -CH2NH-), 1.46 (s,
9H, -CH3).
General procedure for the synthesis of 21a-b. To a round-bottom flask equipped with a stir bar was added
Boc-protected amine 20a-b (1.0 eq), followed by 4 M HCl in 1,4-dioxane (20 eq). The reaction mixture was
stirred for 2 h and concentrated in vacuo. The resulting residue was suspended in a mixture of ethanol and tert-
butyl methyl ether (V/V, 3/1), and the resulting solid was filtered to give the white solid product.
(4-(5-(4-(2-Fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)phenyl)methanamine hydrochl-
1
oride (21a). Yield: 77%. MP: 276-277 °C. H NMR (400 MHz, DMSO-d6) δ 8.71 (s, 2H, -NH2), 8.39 (d, J =
8.4 Hz, 1H, -ArH), 8.29 (s, 1H, -ArH), 8.10 (d, J = 7.6 Hz, 2H, -ArH), 7.73 (d, J = 7.6 Hz, 2H, -ArH), 7.56 (d, J
= 8.7 Hz, 1H, -ArH), 4.91 – 4.67 (m, 2H, -CH2F), 4.62 – 4.46 (m, 2H, -CH2O-), 4.12 (s, 2H, -CH2-). 13C NMR
(101 MHz, DMSO-d6) δ 174.69, 168.31, 159.97, 138.18, 134.58, 130.23, 127.69, 127.10 (d, J = 5.1 Hz),
126.37, 123.34 (d, J = 273.7 Hz), 118.56 (d, J = 31.3 Hz), 116.24, 115.50, 82.13 (d, J = 168.7 Hz), 69.27 (d, J =
19.2 Hz), 42.24. HRMS (ESI) calcd for C18H15F4N3O2 [M + H+] 382.1173. Found [M + H+] 382.1169.
2-((4-(5-(4-(2-Fluoroethoxy)-3-(trifluoromethyl)phenyl)-1,2,4-oxadiazol-3-yl)benzyl)oxy)ethan-1-amine
(21b). Yield: 57%. MP: 266-267 °C. 1H NMR (400 MHz, Acetone-d6) δ 8.47 – 8.35 (m, 2H, -ArH), 8.13 – 8.02
(m, 2H, -ArH), 7.65 – 7.49 (m, 3H, -ArH), 4.87 (d, J = 47.8 Hz, 2H, -CH2F), 4.61 (d, J = 28.7 Hz, 2H, -CH2O-),
13
3.89 (s, 2H, -ArCH2O-), 3.71 – 3.57 (m, 2H, -CH2O-), 2.78 – 2.66 (m, 2H, -CH2NH2). C NMR (101 MHz,
Acetone-d6) δ 174.27, 168.67, 159.90, 145.17, 133.73, 128.82, 128.49, 127.16, 127.08, 126.86 (q, J = 5.1 Hz),
123.15 (d, J = 273.7 Hz), 119.18 (q, J = 31.3 Hz), 114.43, 81.54 (d, J = 169.7 Hz), 68.91 (d, J = 31.3 Hz), 60.86,
52.94, 51.25. HRMS (ESI) calcd for C20H19F4N3O3 [M + H+] 426.1435. Found [M + H+] 426.1432.
4.3. In vitro S1PRs binding assay
[32P]S1P was firstly prepared by incubating sphingosine and [γ-32P]ATP with sphingosine kinase 1 as we
previously reported [20]. [32P]S1P was dissolved in DMSO, and then diluted in the assay buffer (50 mM
HEPES-Na with 5 mM MgCl2, 1 mM CaCl2, and 0.5% fatty acid-free bovine serum albumin, pH = 7.5).
25

ACCEPTED MANUSCRIPT
Compounds were dissolved in DMSO and diluted to different concentrations with assay buffer, followed by
adding commercial cell membranes expressing recombinant human S1PRs (1, 2, 3, 4, and 5) in the assay buffer
at room temperature in 96-well plate. [32P]S1P solution was then added to give a final volume of 150 µL
containing 0.1 nM of [32P]S1P and 1 µg of membrane protein per well. Competitive binding was performed for
60 min at room temperature and terminated by collecting the membranes onto 96-well glass fiber (GF/B)
filtration plates (Millipore, Billerica, MA). Each filter was washed with 200 µL of assay buffer for five times.
The filter bound radionuclide was measured by a Beckman LS3801 scintillation counter using Cherenkov
counting. The reported IC50 values were calculated using the 4 parameter equation, least-square non-linear
regression curve-fit, with GraphPad Prism software (GraphPad Software, Inc). Each assay was repeated at least
three times with duplicate wells for each compound; the reported values (mean ± SD, nM) are calculated from
the average of all assays. Assays for compounds which showed no activity (IC50 > 1000 nM) were only repeated
twice.
4.4. Radiochemistry
4.4.1. Synthesis of precursor 25
4-(3-(4-(Hydroxymethyl)phenyl)-1,2,4-oxadiazol-5-yl)-2-(trifluoromethyl)phenol (22) [18]. To a round-
bottom flask equipped with a stir bar was added 4-hydroxy-3-(trifluoromethyl)benzoic acid (1.96 g, 9.51 mmol),
HOBt (0.26g, 1.90 mmol), TBTU (3.05 g, 9.51 mmol), DMF (20 mL), and DIPEA (3.69 g, 28.5 mmol). The
reaction mixture was stirred for 0.5 h before adding 8e (1.58 g, 9.51 mmol). The reaction mixture was stirred for
1 h at room temperature and refluxed in a pre-heated 120 °C oil-bath. The reaction mixture was stirred for 4 h
and monitored by TLC. After cooling, the reaction mixture was diluted with water and extracted with ethyl
acetate. The ethyl acetate layer was washed with 1 M HCl, saturated brine, and dried over anhydrous MgSO4.
After filtration and concentration, the residue was purified on a silica gel column. (1.43 g, 45%)
4-(3-(4-(Azidomethyl)phenyl)-1,2,4-oxadiazol-5-yl)-2-(trifluoromethyl)phenol (23). To a round-bottom flask
equipped with a stir bar was added 22 (1.39 g, 4.13 mmol), diphenyl phosphoryl azide (1.37 g, 4.96 mmol), and
toluene (10.0 mL). The mixture was cooled to 0 °C before adding 1,8-Diazabicyclo[5.4.0]undec-7-ene (0.76,
26

ACCEPTED MANUSCRIPT
4.96 mmol) dropwise. The reaction was warmed to room temperature slowly and stirred overnight. Then, the
reaction mixture was diluted with water and extracted with ethyl acetate. The ethyl acetate layer was washed
with 1 M HCl, saturated brine and dried over anhydrous MgSO4. After filtration and concentration, the residue
1
was purified on a silica gel column to give yellow solid (815 mg, 55%). MP: 158-161°C. H NMR (400 MHz,
CDCl3) δ 8.41 (s, 1H, -ArH), 8.22 (d, J = 8.5 Hz, 1H, -ArH), 8.17 (d, J = 7.8 Hz, 2H, -ArH), 7.47 (d, J = 7.9 Hz,
2H, -ArH), 7.09 (d, J = 8.6 Hz, 1H, -ArH), 4.44 (s, 2H, -CH2-).
2-(4-(3-(4-(Azidomethyl)phenyl)-1,2,4-oxadiazol-5-yl)-2-(trifluoromethyl)phenoxy)ethyl 4-methylbenzene-
sulfonate (24). To a round-bottom flask equipped with a stir bar was added 23 (500 mg, 1.38 mmol), ethylene
ditosylate (767 mg, 2.07 mmol), K2CO3 (953 mg, 6.90 mmol), and CH3CN (10.0 mL). The reaction mixture
was refluxed in a pre-heated 90 °C oil-bath for 12 h. After cooling, the reaction mixture was diluted with water
and extracted with ethyl acetate. The ethyl acetate layer was washed with saturated brine and dried over
anhydrous MgSO4. After filtration and concentration, the residue was purified on a silica gel column to give
white solid (300 mg, 39%). MP: 169-170 °C. 1H NMR (400 MHz, CDCl3) δ 8.44 (s, 1H, -ArH), 8.33 (d, J = 8.7
Hz, 1H, -ArH), 8.19 (d, J = 8.2 Hz, 2H, -ArH), 7.81 (d, J = 8.3 Hz, 2H, -ArH), 7.48 (d, J = 8.1 Hz, 2H, -ArH),
7.38 – 7.32 (m, 2H, -ArH), 7.10 (d, J = 8.7 Hz, 1H, -ArH), 4.45 (s, 2H, -ArCH2-), 4.43 – 4.38 (m, 4H, -
OCH2CH2O-), 2.45 (s, 3H, -CH3).
2-(4-(3-(4-((4-(hydroxymethyl)-1H-1,2,3-triazol-1-yl)methyl)phenyl)-1,2,4-oxadiazol-5-yl)-2-(trifluorome-
thyl)phenoxy)ethyl 4-methylbenzenesulfonate (25). To a round-bottom flask equipped with a stir bar was
added azide 24 (243 mg, 0.43 mmol) and THF (2.0 mL). Once the azide was dissolved, water (1.1 mL) was
added followed by 3-(methoxymethoxy)prop-1-yne (46 mg, 0.46 mmol), sodium ascorbate (8.5 mg, 0.043
mmol), and copper sulfate pentahydrate (6.4 mg, 0.026 mmol). The reaction mixture was stirred for 4 h and
monitored by TLC. Then, the reaction mixture was diluted with water and extracted with ethyl acetate. The
ethyl acetate layer was washed with saturated brine and dried over anhydrous MgSO4. After filtration and
concentration, a white solid was obtained (100 mg, 35%). MP: 168-171°C. 1H NMR (400 MHz, CD3OD) δ 8.35
– 8.29 (m, 2H, -ArH), 8.11 (d, J = 7.2 Hz, 2H, -ArH), 8.02 (s, 1H, -ArH), 7.74 (d, J = 7.3 Hz, 2H, -ArH), 7.45 (d,
J = 7.5 Hz, 2H, -ArH), 7.36 (d, J = 8.0 Hz, 2H, -ArH), 7.31 (d, J = 8.4 Hz, 1H, -ArH), 5.67 (s, 2H, -ArCH2-),
27

ACCEPTED MANUSCRIPT
4.65 – 4.61 (m, 4H, -CH2OCH2O-), 4.43 – 4.34 (m, 4H, -OCH2CH2O-), 3.27 (s, 3H, -OCH3), 2.37 (s, 3H, -
ArCH3).
4.4.2. Radiosynthesis of [18F]12b
[18F]KF (~7.4 GBq) aqueous was added to a vial containing Kryptofix 222 (~6 mg), and dried by azeotropic
evaporation with CH3CN (3 x 1 mL) under N2 flow at 110 °C. To the reaction vial was added precursor 25 (2
mg) as a solution in CH3CN (300 µL). The reaction was placed in a 110 °C oil-bath for 15 min. The reaction
was removed from the oil-bath, at which time 6 M HCl (150 µL) was added. The reaction mixture was heated in
a 110 °C oil-bath for another 5 min. The reaction was removed from the oil-bath and quenched with 6 M NaOH
(150 µL) and diluted with 2.4 mL of the HPLC mobile phase (45% CH3CN in 0.1 M ammonium formate buffer,
pH = 4.5). The mixture was passed through a Sep-Pak® Alumina N Cartridge (Part No. WAT020510) and
injected onto the semi-preparation HPLC column (Agilent SB-C18 250 x 9.6 mm, 5 µm, UV = 254 nm, 4.0
mL/min). The retention time of [18F]12b is around 19-22 min. The HPLC fraction was collected into a water
bottle with 60 mL of water and then trapped on a Sep-Pak® C-18 Cartridge (Part No. WAT020515). The
activity (~740 MBq) was washed out with 0.6 mL of ethanol and 5.4 mL of 0.9% saline. After sterile filtration
into a glass vial, the [18F]12b was ready for quality control (QC) analysis and animal studies. An aliquot of
sample was injected onto an analytical HPLC to determine the concentration of tracer. Meanwhile, the tracer
was authenticated by co-injected with non-radiolabeled standard 12b sample solution. The HPLC condition are
as follows: Agilent Zorbax SB-C18 column (250 × 4.6 mm); UV absorbance at 254 nM; the mobile phase is
consisted of 45% CH3CN in 0.1 M ammonium formate buffer, pH = 4.5; the flow rate is 1.0 mL/min; the
retention time of [18F]12b is 5.7 min.
4.5. Ex vivo autoradiography study
An adult male SD rat (~450 g, Charles River) was injected with [18F]12b (~55 MBq) via the tail vein under 2–
3% isoflurane/oxygen anesthesia and euthanized at 60 min post-injection. The brain was immediately removed
and snap-frozen over dry ice followed by sectioning at 100 µm and mounting on glass slides. Frozen slides were
directly exposed to a BAS storage phosphor screen film (BAS-IP-MS-2025) in an imaging cassette (Fuji Photo
28