Design, synthesis, and in vitro bioactivity evaluation of fluorine-containing analogues for sphingosine-1-phosphate 2 receptor

Article abstract of DOI:10.1016/j.bmc.2019.06.047

Twenty eight new aryloxybenzene analogues were synthesized and their in vitro binding potencies toward S1PR2 were determined using a [³²P]S1P competitive binding assay. Out of these new analogues, three compounds, 28c (IC₅₀ = 29.9 ± 3.9 nM), 28e (IC₅₀ = 14.6 ± 1.5 nM), and 28g (IC₅₀ = 38.5 ± 6.3 nM) exhibited high binding potency toward S1PR2 and high selectivity over the other four receptor subtypes (S1PR1, 3, 4, and 5; IC₅₀ > 1000 nM). Each of the three potent compounds 28c, 28e, and 28g contains a fluorine atom that will allow to develop F-18 labeled PET radiotracers for imaging S1PR2.

Full text of DOI:10.1016/j.bmc.2019.06.047

Accepted Manuscript
Design, synthesis, and in vitro bioactivity evaluation of fluorine-containing an-
alogues for sphingosine-1-phosphate 2 receptor
Zonghua Luo, Hui Liu, Robyn S. Klein, Zhude Tu
PII:
S0968-0896(19)30732-1
https://doi.org/10.1016/j.bmc.2019.06.047
BMC 14984
DOI:
Reference:
Bioorganic & Medicinal Chemistry
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Received Date:
Revised Date:
Accepted Date:
7 May 2019
18 June 2019
28 June 2019
Please cite this article as: Luo, Z., Liu, H., Klein, R.S., Tu, Z., Design, synthesis, and in vitro bioactivity evaluation
of fluorine-containing analogues for sphingosine-1-phosphate 2 receptor, Bioorganic & Medicinal Chemistry
(2019), doi: https://doi.org/10.1016/j.bmc.2019.06.047
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Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com
Design, synthesis, and in vitro bioactivity evaluation of fluorine-containing analogues
for sphingosine-1-phosphate 2 receptor
Zonghua Luoa , Hui Liua , Robyn S. Kleinb,c,d , and Zhude Tua, *
a Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110, USA
b Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA
c Department of Neuroscience, Washington University School of Medicine, St. Louis, MO 63110, USA
d Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, MO 63110, USA
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
Twenty eight new aryloxybenzene analogues were synthesized and their in vitro binding
potencies toward S1PR2 were determined using a [32P]S1P competitive binding assay. Out of
these new analogues, three compounds, 28c (IC50 = 29.9 ± 3.9 nM), 28e (IC50 = 14.6 ± 1.5 nM),
and 28g (IC50 = 38.5 ± 6.3 nM) exhibited high binding potency toward S1PR2 and high
selectivity over the other four receptor subtypes (S1PR1, 3, 4, and 5; IC50 > 1000 nM). Each of
the three potent compounds 28c, 28e, and 28g contains a fluorine atom that will allow
developing F-18 labeled PET radiotracers for imaging S1PR2.
Keywords:
Sphingosine 1-phosphate receptor 2
Binding potency
2009 Elsevier Ltd. All rights reserved.
Selectivity
Positron emission tomography
(MS) patients with compared to their male counterparts.17 In the
kidney of diabetic rats and mesangial cells under high glucose
condition, the highest S1PR2 mRNA expression was observed
1. Introduction
Sphingosine 1-phosphate (S1P) is an essential membrane-
derived bioactive sphingolipid mediator which plays critical
regulatory functions in the cardiovascular, nervous, and immune
systems.1 S1P is generated from the phosphorylation of
sphingosine by sphingosine kinases (Sphk1 and 2) and presents
at high levels in the blood and lymph.2 S1P exerts its functions by
interacting with a family of five G protein-coupled receptor
subtypes, S1PR1, 2, 3, 4, and 5, originally named as EDG-1, 5, 3,
6, and 8.3 Each subtype has its specific tissue expression and
compared to other four S1P subtypes, S1PR1, 3, 4, and 5.18
Together, S1PR2 protein may serve a promising biomarker for
assessing the progress of diseases such as cholangiopathies, MS,
and diabetic nephropathy. Positron emission tomography (PET)
is a unique imaging modality and can provide a non-invasive
method to quantitatively assess the target protein distribution in
vivo in tissues. It was widely used in preclinical and clinical
investigations. PET imaging with a suitable S1PR2 specific
radiotracer could help advance our understanding of S1PR2
modulating functions in related diseases. Therefore, identification
response to different biological process.4, Out of these five
5
subtypes, S1PR1, 2, and 3 are extensive ubiquitously expressed
in a variety of cell types and tissues; S1PR4 expression is
primarily in hematopoietic and immune system cells; S1PR5 is
primarily expressed in the spleen and central nervous system
(CNS).6, 7 Among these receptors, S1PR2 plays a pivotal role in
mediating different cellular functions and pathologies, such as
CN
O
N
H
H
N
N
N
Cl
N
N
N
N
H
O
N
O
Cl
JTE-013
CYM-5520
OH
endothelial,8 metabolic,9, muscle,11 neuronal,12, and kidney
functions.14 Conjugated bile acid induced activation of the S1PR2
signaling pathway plays a critical role in obstructive cholestasis
and invasive growth of esophageal adenocarcinoma (EAC) cells,
which represent a novel therapeutic target for cholestatic liver
10
13
H
N
N
O
11CH3
O
H
H
N
O
N
N
F
N
N
N
H
R
O
N
Cl
1, R = H
2, R=
3, R=
COOH
O
diseases and EAC.15, During demyelination process in CNS
16
11
TZ34125
C]
[
CH2OOH
diseases, S1PR2 regulates blood-brain barrier (BBB) function
and an increased expression of S1PR2 is observed in disease-
susceptible regions of both female SJL experimental allergic
encephalomyelitis (EAE) mice and female multiple sclerosis
4, R=
N
O
O
Figure 1. Structures of S1PR2 compounds

of S1PR2 specific ligands is imperative. Despite recognition of
this potential, only a few compounds have been reported
specifically for S1PR2 (Figure 1). JTE-013, a well-known
antagonist for S1PR2, is widely used in preclinical studies19;
CYM-5520 is an allosteric S1PR2 selective agonist, but it was
not competitive with S1P in a [33P]S1P competitive binding
assay.20 Takuya Seko’s group recently, reported four potent
S1PR2 compounds 1-4 (Figure 1) that have high antagonist
activity. Compounds 2, 3, and 4 showed improved metabolic
stability compared to compound 1.21-23 Our group reported the
radiosynthesis of [11C]TZ34125, a JTE-013 analogue, and
performed in vivo evaluation in SJL mice that confirmed the
sexual dimorphism of S1PR2 expression in the cerebellum.24
Nevertheless, no F-18 S1PR2 radiotracer was reported. To
identify an F-18 PET radiotracer for imaging S1PR2 expression
in vitro and in vivo for related diseases, herein we reported our
recent efforts on the exploration of the new structural S1PR2
ligands.
After analyzing the structure of compound 1, it was dissected
into three fragments A, B, and C as shown in Figure 2. Our
exploration of new S1PR2 analogues used three strategies. First,
we focused on the optimization of fragment A: the proton at the
5-position of benzyl ring was replaced with different moieties
including alkoxy heterocycle, trifluoromethyl, aryloxy, and
arylthio groups. Second, we focused on the optimization of
fragment B: different electronegativity functional groups were
used to replace the fluorine at 4’-position of the aromatic ring.
Third, we focused on the optimization of fragment C: the
piperdin-4-ol was replaced with pyrrolidin-3-ol and azetidin-3-ol
and different alkyl chains were used to replace the 2-(ethyl)butyl
tail. The goal of our efforts was to identify S1PR2 ligands with
high binding potency and high selectivity. The newly synthesized
analogues were screened for their in vitro binding potency and
selectivity using [32P]S1P as the competitive radioligand in an
assay with cell membranes that were enriched for the different
receptor subtypes. Out of these new analogues, three new
compounds were discovered having high S1PR2 binding potency
(IC50 < 50 nM) and high selectivity for S1PR2. Consequently, the
structure-activity relationship analysis of these new analogues
was explored.
OH
a, b
Cbz
O2N
N
O
HN
5
6
O2N
O
O2N
F
F
d
c
e
F
O
O
F
F
F
n
n
7
8a
8b
: n = 1; : n = 2
9a
9b
: n = 1; : n = 2
OH
H
H
Cl3C
O
N
O
N
N
O
f
O
O
F
F
O
O
F
n
F
n
10a
10b
11a
11b
: n = 1; : n = 2
: n = 1;
: n = 2
Scheme 1. Synthesis of 11a-b. Reagents, conditions, and yields: (a)
LaCl3-2LiCl solution, 2-ethylbutylmagnesium bromide, 0 ºC - RT; (b)
Pd/C, H2, methanol, RT; 81%; (c) 2-fluorethanol or 3-fluoropyopan-1-
ol, NaH, DMF, RT; 33-35%; (d) 4-fluorophenol, K3PO4, DMA, 100 ºC;
91-99%; (e) (i) Pd/C, H2, ethyl acetate, RT; (ii) 2,2,2-trichloroethyl
chloroformate, ethyl acetate, NaHCO3, 0 ºC – RT; (f) 6, DIPEA, DMA,
100 ºC; 97-98%.
lanthanum trichloride-lithium chloride complex at 0 ºC to afford
alcohol intermediate, following by removal of carboxybenzyl
(Cbz) group through Pd-catalyzed hydrogenation in methanol to
yield
4-(2-
ethylbutyl)piperidin-4-ol
(6).
Meanwhile,
commercially available 1,3-difluoro-5-nitrobenzene (7) was
treated with 2-fluorethanol or 3-fluoro-1-propanol and sodium
hydride (NaH) in N,N-dimethylformamide (DMF) to yield 8a-b,
which were subjected to a substitution reaction using 4-
fluorophenol to afford aryloxy intermediates 9a-b in the presence
of K3PO4 at 100 ºC in N,N-dimethylacetamide (DMA). The Pd-
catalyzed hydrogenation of 9a-b gave anilines, followed by
treating with 2,2,2-trichloroethyl chloroformate and NaHCO3
gave intermediates 10a-b. The subsequent coupling reaction of
10a-b with 4-(2-ethylbutyl)piperidin-4-ol (6) in the presence of
N,N-diisopropylethylamine (DIPEA) at 100 ºC in DMA afforded
target compound 11a-b.
The synthesis of 17a-c were achieved by following Scheme 2.
Commercially available 4-fluorophenol (12) reacted with 1-
fluoro-3-nitro-5-(trifluoromethyl)benzene or 1,3-difluoro-5-
nitrobenzene afforded 13 or 14, respectively. Intermediate 14 was
coupled with 1-methylpiperozine or 1H-pyrazole using K2CO3 as
a base in dimethyl sulfoxide (DMSO) to afford 15a or 15b. After
palladium-catalyzed reduction of 13 and 15a-b, the resulting
anilines were reacted with 2,2,2-trichloroethyl chloroformate to
afford 16a-c. The target compounds 17a-c was prepared by
reacting the carbamates 16a-c with 4-(2-ethylbutyl)piperidin-4-ol
(6) as described for 11a-b.
OH
alkyl
N
C
OH
A
H
2
5
B
1'
O
N
N
4'
F
O
substituted group
OH
alkyl
alkoxy
heterocycle
CF3
aryloxy group
arylthiothio group
N
n
Secondly, to explore the impact of the substitution group at 4’-
position of the aromatic ring in fragment B, we retained the
trifluoromethyl group because trifluoromethyl-containing
compound 17c showed moderate binding potency. Compounds
21a-f were synthesized with different substitution groups at 4’-
position as shown in Scheme 3. Briefly, commercially available
1- fluoro-3-nitro-5-(trifluoromethyl)benzene (18) reacted with
differently substituted phenols in the presence of K3PO4 in DMA
afforded 19a-f. The palladium-catalyzed hydrogenation of 19a-f
gave anilines, followed by condensation with 2,2,2-trichloroethyl
chloroformate to afford key intermediates 20a-f. Coupling of
intermediates 20a-f with 4-(2-ethylbutyl)piperidin-4-ol (6)
yielded the target compounds 21a-f.
Figure 2. Design strategy of new S1PR2 analogues
2. Results and Discussion
2.1 Chemistry
Our exploration of new S1PR2 analogues was accomplished
by following our abovementioned three strategies. Firstly,
fluorine substituted alkoxy, 1-methyl-4-piperazine, 1H-pyrazole,
and trifluoromethyl groups was used to replace the hydrogen
atom at the 5-position of the aromatic ring in the fragment A to
generate compounds 11a-b and 17a-c. Briefly, the synthesis of
compounds 11a-b were accomplished by following Scheme 1.
Briefly, benzyl 4-oxopiperidine-1-carboxylate (5) was treated
with 2-ethylbutylmagnesium bromide in the presence of

O2N
O
OH
R1
O2N
O
a, b
c
Cbz
N
O
HN
d
F
F
F
R
1
5
22a
: R = methyl;
1
: R1 = ethyl
22b
H
N
N
N
Cl3C
O
N
O
: R = isopropyl;
: R1 = isobutyl
14
15a
: R =
22c
22d
15b
: R =
O
OH
F
N
b
a, b
R
HN
Cbz
N
O
n
n
N
N
N
16a
16b
16c
: R =
O2N
O
23a
23b
24a
24b
: n = 2
, n = 1;
, n = 2
: n = 1;
F
: R =
: R =
a
d
N
F
OH
CF3
OH
CF3
13
R1
H
N
12
Cl3C
O
O
H
N
c
N
O
O
R2
O
R2
OH
CF3
N
N
N
CF3
25a-h
H
17a
17b
17c
: R =
N
O
N
O
e
H
N
: R =
: R =
2
2
S
20c
20f
: R
: R
=
=
O
N
O
F
O
H
N
CF3
R2
=
S
R2
=
R
17a-c
O O
O
1 = -CH3
25b,
25d,
25f,
R
R
1 = -CH3
25a,
R
R
R
R
1 = -CH2CH3
1 = -CH2CH3
25c,
25e,
25g,
Scheme 2. Synthesis of 17a-c. Reagents, conditions, and yields: (a)
1-fluoro-3-nitro-5-(trifluoromethyl)benzene, K3PO4, DMA, 100 ºC;
93%; (b) 1,3-difluoro-5-nitrobenzene, K3PO4, DMA, 100 ºC; 95%;
(c) 1-methylpiperozine or 1H-pyrazole, K2CO3, DMSO, 65 ºC; 42-
53%; (d) (i) Pd/C, H2, ethyl acetate, RT; (ii) 2,2,2-trichloroethyl
chloroformate, ethyl acetate, NaHCO3, 0 ºC - RT; (e) 6, DIPEA,
DMA, 100 ºC ; 22-44%.
1 = -CH(CH3)2
1 = -CH2CH(CH3)2
R1 = -CH(CH3)2
1 = -CH2CH(CH3)2
25h,
R
OH
H
N
Cl3C
O
O
H
N
N
O
c
H
N
n
O
H
N
O
H
CF3
20c
O
Cl3C
O
N
O
CF3
25j
O
O2N
O
O2N
F
25i
: n = 1;
Scheme 4. Synthesis of different amines 22a-d, 24a-b, : ann=d2 target
compounds 25a-j. Reagents, conditions, and yields: (a) LaCl3-2LiCl
solution, Grignard's agents, 0 ºC - RT; (b) Pd/C, H2, methanol, RT;
92-98%; (c) amines (22a-d or 24a-b), DIPEA, DMA, 100 ºC; 42-
99%.
a
b
O
R
R
CF3
CF3
19a
CF3
18
-f
20a
-f
OH
ketones. The subsequent coupling two carbamates 20c and 20f
with corresponding amines (22a-d and 24a-b) afforded
compounds 25a-j.
N
O
H
N
21a
O
21d
21e
: R =
: R =
: R =
: R =
: R =
: R =
N
O
O
c
O
NH2
21b
21c
O
O
R
O
S
H
N
21f
CF3
Finally, to further investigate the substituted group at the 5-
position of the middle aromatic ring in fragment A, a series of
substituted aryloxy group and arylthio group were used to replace
the trifluoromethyl group. New analogues 28a-g were
synthesized by following Scheme 5. Briefly, compound 14
reacted with 4-methoxylphenol, 6-hydroxy-2-methyl-3,4-
dihydroisoquinolin-1(2H)-one, 6-hydroxy-3,4-dihydroisoquino-
lin-1(2H)-one, N-(2-fluoroethyl)-4-hydroxybenzamide, or 4-
(methylsulfonyl)benzene-thiol in the presence of K3PO4 gave
O
O
21a-f
Scheme 3. Synthesis of target compounds 21a-f. Reagents, conditions,
and yields: (a) phenols, K3PO4, DMA, 100 ºC; 60-99%; (b) (i) Pd/C,
H2, ethyl acetate, RT; (ii) 2,2,2-trichloroethyl chloroformate, ethyl
acetate, NaHCO3, 0 ºC - RT; (c) 6, DIPEA, DMA, 100 ºC; 39-81%.
Thirdly, to further evaluate the impact of the side chain and N-
containing heterocycle in fragment C, we changed 2-(ethyl)butyl
side chain with different alkyl chains and replaced the piperdin-4-
ol with pyrrolidin-3-ol and azetidin-3-ol. Compounds 25a-j were
synthesized by following Scheme 4. The amines (22a-d and 24a-
b) were prepared ahead using the same procedure as described
for compound 6 with corresponding Grignard's agents and
compounds
26a-e,
followed
by
palladium-catalyzed
hydrogenation afforded corresponding anilines. The resulting
anilines were treated with 2,2,2-trichloroethyl chloroformate and
NaHCO3 to afford intermediates 27a-e. The subsequent
H
O2N
O
O2N
O
Cl3C
O
N
O
a
b
c
O
F
F
F
R1
R1
F
14
26a-e
27a-e
S
O
S
O
1
1
1
R2 = 2-(ethyl)butyl;
R2 = 2-(ethyl)butyl;
R2 = 2-(ethyl)butyl;
R2 = 2-(ethyl)butyl
: R
=
=
=
R2 = 2-(ethyl)butyl
R2 = isobutyl
28a
28e
28f
: R
=
=
=
=
O
S
O
OH
R2
O
H
N
N
O
O
O
1
28b
28c
28d
: R
: R
H
N
N
F
O
O
F
O
R1
28a-g
1
1
1
R2 = isobutyl
28g
: R
: R
O
S
O
NH
O
O
H
N
: R
F
O
Scheme 5. Synthesis of 28a-g. Reagents, conditions, and yields: (a) phenols or thiophenol, K3PO4, DMA, 100 ºC; 15-99%;
(b) (i) Pd/C, H2, ethyl acetate, RT; (ii) 2,2,2-trichloroethyl chloroformate, ethyl acetate,NaHCO3, 0 ºC - RT; 90-99%; (c) 6 or
22d, DIPEA, DMA, 100 ºC; 24-88%.

condensation reaction of 27a-e with 6 or 22d in the presence of
DIPEA at 100 ºC in DMA yielded target compounds 28a-g.
binding data indicated that mono-methyl carbamide (CONHCH3)
and methylsulfonyl (SO2CH3) group improved the binding
activity, compound 21c and 21f had IC50 values of 278.5 and
270.3 nM, respectively. The other functional groups, like OCH3,
CONH2, CON(CH3)2, and COOCH3 in 21a, 21b, 21d, and 21e
decreased the binding activity with IC50 >1000 nM. To
investigate the impact of the side chain in the fragment C,
compounds 25a-h were synthesized and tested. We observed that
the replacement of 2-(ethyl)butyl chain with methyl, ethyl, or
isopropyl chains caused the loss of biological activity,
compounds 25a-f had IC50 > 1000 nM. Interestingly, we
2.2 Biological binding studies
In vitro binding of these newly synthesized compounds 11a-b,
17a-c, 21a-f, 25a–j, and 28a–g toward S1PR2 was determined by
radioligand [32P]S1P competitive cell membrane binding assay
following our published protocol.20 The results are shown in
Table 1-2. The strategy of introducing alkoxy, heterocycle, or
trifluoromethyl group in fragment A gave compounds 11a-b and
17a-c. The in vitro S1PR2 binding data showed that compound
17c, with a trifluoromethyl group at the 5-position exhibit
moderate binding activity with an IC50 value of 362.3 nM, which
is comparable to the lead compound 1 having IC50 value of 310
nM for S1PR2. The fluorine-containing alkoxy compounds, 11a
and 11b showed low binding activities with IC50 > 1000 nM. The
substitution with another two N-containing heterocycles, 1-
methyl-4-piperazine and 1H-pyrazole didn’t improve the binding
activity either; both 15a and 15b had IC50 > 1000 nM.
Subsequently, our further exploration of new analogues focused
on structural optimization of compound 17c. From one side, as
shown in Scheme 3, we first retained trifluoromethyl group at 5-
position in the fragment A and checked the impact of various
substituted groups at 4’-position in fragment B (21a-f). Our
observed compound 25g (IC50 = 359.3 nM) and 25h (IC50
=
296.5 nM) with a isobutyl side chain resulted in comparable
binding activity compared to the 2-(ethyl)butyl compounds 21c
and 21f. It suggested that the size and the steric hindrance of the
side chain lead to increase the S1PR2 binding activity. Another
strategy to modify the fragment C was to change the piperidine
heterocycle moiety. The results revealed that the compounds 25i
and 25j, with pyrrolidine and azetidine heterocycles,
respectively, showed IC50 values that were over 1000 nM, which
indicated the piperidine heterocycle was the favorable
heterocycle moiety. From the other side, compound 2, having a
4-pyridineoxyl moiety showed potent binding activity with an
IC50 value of 45 nM, we replaced the trifluoromethyl group at 5-
position with different alkoxy moieties in fragment A and
Table 1 Structures and binding potencies (IC50 ± SD) of compounds 11a-b, 17a-c, 21a-f, and 25a-j toward S1PR2. a
OH
OH
n
R2
R2
H
H
N
N
O
N
N
O
O
R3
O
R3
R1
R1
1, 11a-b, 17a-c, 21a-f, and 25a-h
25i-j
Compd.
JTE-013
1b
n
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
1
2
R1
-
R2
R3
-
S1PR2 IC50 (nM)
66.8 ± 7.8
310
-
H
2-(ethyl)butyl
2-(ethyl)butyl
2-(ethyl)butyl
2-(ethyl)butyl
2-(ethyl)butyl
2-(ethyl)butyl
2-(ethyl)butyl
2-(ethyl)butyl
2-(ethyl)butyl
2-(ethyl)butyl
2-(ethyl)butyl
2-(ethyl)butyl
methyl
F
F
F
F
F
F
11a
11b
17a
17b
17c
OCH2CH2F
> 1000
O(CH2)3F
> 1000
1-methylpiperazine
> 1000
1H-pyrazole
CF3
> 1000
362.3 ± 85.0
> 1000
21a
21b
21c
CF3
OCH3
CF3
CONH2
> 1000
CF3
CONHCH3
CON(CH3)2
COOCH3
SO2CH3
278.5 ± 27.2
> 1000
21d
21e
CF3
CF3
> 1000
21f
CF3
270.3 ± 72.0
> 1000
25a
25b
25c
CF3
CONHCH3
SO2CH3
CF3
methyl
> 1000
CF3
ethyl
CONHCH3
SO2CH3
> 1000
25d
25e
CF3
ethyl
> 1000
CF3
isopropyl
CONHCH3
SO2CH3
> 1000
25f
CF3
isopropyl
> 1000
25g
25h
25i
CF3
isobutyl
CONHCH3
SO2CH3
359.3 ± 50.1
296.5 ± 32.3
> 1000
CF3
isobutyl
CF3
isobutyl
CONHCH3
CONHCH3
25j
CF3
isobutyl
> 1000
a IC50 values were determined by at least two independent experiments, each run was performed in duplicate. b reference21

Table 2 Structures and binding affinities (IC50 ± SD) of 28a-g toward S1PRs.a
OH
R2
H
N
N
O
O
F
R1
2 and 28a-g
IC50 (nM)
S1PR3
Compd.
R1
R2
S1PR1
> 1000
S1PR2
66.8 ± 7.8
3.6 ± 0.5
S1PR4
> 1000
S1PR5
> 1000
JTE-013
S1Pb
-
-
-
-
> 1000
1.4 ± 0.3
0.4 ± 0.2
151 ± 82
3.1 ± 1.1
O
2c
2-(ethyl)butyl
2-(ethyl)butyl
> 10000
N.T.
45
> 10000
N.T.
> 10000
N.T.
> 10000
N.T.
N
O
28a
188.5 ± 32.9
O
O
N
28b
2-(ethyl)butyl
> 1000
73.3 ± 10.6
> 1000
> 1000
> 1000
O
O
NH
28c
28d
28e
28f
2-(ethyl)butyl
2-(ethyl)butyl
2-(ethyl)butyl
isobutyl
> 1000
> 1000
> 1000
N.T.
29.9 ± 3.9
66.7 ± 7.8
14.6 ± 1.5
194.5 ± 35.9
38.5 ± 6.3
> 1000
> 1000
> 1000
N.T.
> 1000
> 1000
> 1000
N.T.
> 1000
> 1000
> 1000
N.T.
O
O
H
N
F
O
S
O
S
O
O
H
N
F
O
S
O
S
28g
isobutyl
> 1000
> 1000
> 1000
> 1000
O
a
IC50 values were determined by at least two independent experiments, each run was performed in duplicate; for compounds with IC50 < 100 nM, at least three
independent experiments were performed, each run was performed in duplicate; N.T. means not test; b reference25; c reference21
identified compounds 28a-g. The piperdine heterocycle moiety
with isobutyl or 2-(ethyl)butyl side chain was retained because
these two side chains were discovered as favorable
pharmacophores in current analogues. As shown in Table 2, the
aryloxy moieties containing compounds exhibited improved
S1PR2 binding activity compared to compound 17c. Compound
28a showed an IC50 value of 188.5 nM; compounds 28b and 28c,
bearing a cyclization of carbamide group exhibited good S1PR2
binding potency that compound 28b had an IC50 value of 73.3
nM and compound 28c had an IC50 value of 29.9 nM; Compound
28d possessing a fluoroethyl carbamide group had an IC50 value
binding potency toward S1PR2 with an average IC50 value of
14.6 nM.
From the in vitro binding data shown in Table 1 and Table 2,
the following structure-activity relationship information was
generated: a) when the 5-position substitution groups were
introduced into the fragment A, the binding potency order of the
substituents is aryloxy > CF3 > alkoxyl and N-containing
heterocycles, the aryloxy group play an important role in
regulating the S1PR2 binding activity; b) when the 4’-position
substitution groups were introduced into the fragment B, the
stronger electronegativity of substituents offered the higher
S1PR2 binding activity with the order as SO2CH3 > CONHCH3 >
F > CONH2, CON(CH3)2, COOCH3, OCH3; c) when optimizing
the fragment C, comparing with different group, the piperidine
ring was the most favorable heterocycle and the steric hindrance
of side chains resulted in increased S1PR2 binding potency with
the order as 2-(ethyl)butyl > isobutyl > isopropyl, ethyl, and
methyl. The structure-activity relationship information of this
scaffold is valuable for guiding the future design of new S1PR2
compounds.
of
66.7
nM;
compound
28e,
containing
a
4-
(methylsulfonyl)benzene-thiol ether moiety was the most potent
S1PR2 compound with an IC50 value of 14.6 nM, which is more
potent than the 4-pyridineoxyl lead compound 2 having IC50
value of 45 nM. Additionally, both of compounds 28f (IC50
=
194.5 nM) and 28g (IC50 = 38.5 nM) possessing an isobutyl side
chain exhibited less potency for S1PR2 than the corresponding
28d and 28e, suggesting 2-(ethyl)butyl side chain is the most
favorable moiety for S1PR2 binding activity. The representative
competitive binding curves of compounds JTE-013, 28b, 28c,
28d, 28e, and 28g toward S1PR2 were shown in Figure 3. Six
different concentrations from 0.01 to 1000 nM were used to
determine the binding potencies. As shown in the competitive
binding curves, compound 28e has the best inhibition effect at
1.0 nM compared to the other compounds and it gave the best
Because compounds 28b, 28c, 28d, 28e, and 28g showed high
S1PR2 binding potency with IC50 < 100 nM, their binding
potencies toward the other S1P receptor subtypes, S1PR1, 3, 4,
and 5 were also assessed to determine their in vitro binding
selectivity for S1PR2. As shown in Table 2, all five compounds
had no significant binding toward the other four subtypes

EMD Chemicals Inc. Visualization was accomplished with
ultraviolet light (UV 254 nm), or by shaking the TLC plate in a
sealed jar containing silica gel and iodine. Flash column
chromatography was performed using 230–400 mesh silica gel
purchased from Silicycle. All work-up and purification
procedures were carried out with reagent grade solvents in the
air. Yields refer to isolate yield unless otherwise stated. Melting
points were determined on a MEL-TEMP 3.0 apparatus. 1H NMR
and 13C NMR spectra were recorded on Varian 400 MHz
instrument. Chemical shifts are reported in parts per million
(ppm) and are calibrated using residual undeuterated solvent as
an internal reference (CDCl3: δ 7.26 ppm; CD3OD: δ 3.31 ppm;
DMSO: δ 2.50 ppm). Data are reported as follows: chemical
shift, multiplicity, coupling constants (Hz), and integration. High
resolution positive ion mass (HRMS) analyses were conducted
on a Bruker MaXis 4G Q-TOF mass spectrometer with
electrospray ionization source.
4.1.1 Synthesis of 4-(2-ethylbutyl)piperidin-4-ol (6)
To a dried round two neck bottomed flask equipped with a
magnetic stir bar were added 0.6 M LaCl3-2LiCl in THF (12.5
mL, 7.5 mmol) under nitrogen, 0.25 M 2-ethylbutylmagnesium
bromide in THF (30 mL, 7.5 mmol) was added slowly through
syringe at 0 ºC. After stirring at room temperature for 3 h, a
solution of benzyl 4-oxopiperidine-1-carboxylate (5) (1.2 g, 5.0
mmol) in THF (5.0 mL) was added into the mixture. The reaction
was stirred for another18 h until the reaction was completed as
determined by TLC and then quenched with 25% acetic acid. The
mixture was extracted with ethyl acetate, the ethyl acetate layer
was washed with saturated brine and dried over anhydrous
MgSO4. After filtration and concentration, the crude product was
used directly for the next step without purification.
Figure 3. The representative competitive binding curves of
compounds JTE-013, 28b, 28c, 28d, 28e, and 28g toward S1PR2. The
averaged IC50 values were obtained from three independent
experiments.
S1PR1,3,4, and 5 (IC50 > 1000 nM), indicating that they are
highly selective for S1PR2.
To a round-bottomed flask equipped with a magnetic stir bar
were added above crude product, 10% Pd/C (0.2 g) and methanol
(10.0 mL). The reaction was bubbled with hydrogen gas for 6 h
at room temperature until the reaction was completed as
determined by TLC and then filtered through celite. The filtrate
was concentrated under reduced pressure to afford yellow oil
product 6. Yield: 81%. H NMR (400 MHz, CDCl3) δ 3.18 (s,
1H), 2.98 (d, J = 33.4 Hz, 4H), 1.96 (s, 1H), 1.63 (d, J = 24.9 Hz,
4H), 1.46 – 1.30 (m, 7H), 0.86 (t, J = 7.1 Hz, 6H).
3. Conclusion
In summary, we successfully synthesized a series of ligands
for S1PR2. The in vitro data suggested that compounds 28c, 28e,
and 28g exhibit high S1PR2 binding potencies with IC50 values
of 29.9, 14.6, and 38.5 nM, respectively. They are more potent
for S1PR2 than compound JTE-013 and lead compounds 1 and 2.
In addition, compounds 28c, 28e, and 28g also displayed high
selectivity over S1PR 1, 3, 4, and 5. The initial structure-activity
relationship analysis indicates that aryloxy substitution at 5-
position of fragment A plays a key role in retaining the high
potency for S1PR2; the strong electronegativity substituent at 4’-
position of fragment B is favorable to the S1PR2 binding; the
piperidine ring with steric hindrance side chain in fragment C
also provides S1PR2 favorable binding. Three S1PR2 potent
compounds 28c, 28e, and 28g contain fluorine atom that provides
the position for introducing F-18 isotope to make the F-18
labeling counterparts. Further evaluation of the F-18 labeled
radiotracer may lead to identify an F-18 PET radiotracer for
imaging S1PR2 in vivo. Further characterizations of their in vitro
and in vivo suitability to be PET radiotracers for assessing the
S1PR2 expression in living animals are ongoing in our lab.
1
4.1.2 General procedure to synthesize 8a-b.
A solution of 2-fluoroethanol or 3-fluoropyopan-1-ol (1.0 eq)
o
in DMF (0.25 M) was stirred and cooled to 0 C, then NaH (2.0
eq) was added. After stirring for 15 min, a solution of 1,3-
difluoro-5-nitrobenzene (7) (1.0 eq) in DMF (0.5 M) was added
to the mixture and stirred at room temperature for 12 h until the
reaction was completed as determined by TLC. The reaction then
was diluted with water and extracted with ethyl acetate. The
organic layer was washed with saturated brine and dried over
anhydrous MgSO4. After filtration and concentration, the crude
product was purified on a silica gel column, eluted with
hexane/ethyl acetate to afford 8a-b.
4.1.2.1 1-Fluoro-3-(2-fluoroethoxy)-5-nitrobenzene (8a).
4. Experimental
Compound 8a was eluted with hexane/ethyl acetate (10/1,
1
V/V) as yellow oil. Yield: 33%. H NMR (400 MHz, CDCl3) δ
4.1 Chemistry
7.60 – 7.55 (m, 2H), 6.99 (dt, J = 9.6, 2.3 Hz, 1H), 4.87 – 4.83
(m, 1H), 4.75 – 4.71 (m, 1H), 4.35 – 4.31 (m, 1H), 4.29 – 4.25
(m, 1H).
Commercially available starting materials, reagents, and
solvents were used as received. Unless otherwise indicated, all
reactions were conducted in oven-dried glassware. In general,
anhydrous reactions were performed under nitrogen. Reactions
were monitored by thin-layer chromatography (TLC) carried out
on pre-coated glass plates of silica gel (0.25 mm) 60 F254 from
4.1.2.2 1-Fluoro-3-(3-fluoropropoxy)-5-nitrobenzene (8b)
Compound 8b was eluted with hexane/ethyl acetate (10/1,
1
V/V) as yellow oil. Yield: 35%. H NMR (400 MHz, CDCl3) δ

7.58 – 7.56 (m, 1H), 7.55 – 7.50 (m, 1H), 6.98 – 6.92 (m, 1H),
4.71 (t, J = 5.7 Hz, 1H), 4.59 (t, J = 5.7 Hz, 1H), 4.17 (t, J = 6.1
Hz, 2H), 2.29 – 2.14 (m, 2H).
NMR (101 MHz, CDCl3) δ 158.93 (d, J = 243.4 Hz), 159.90,
159.04, 154.32, 152.31 (d, J = 2.0 Hz), 141.31, 120.89 (d, J = 8.1
Hz), 116.25 (d, J = 24.2 Hz), 102.18, 100.41, 99.87, 81.74 (d, J =
171.7 Hz), 70.17, 67.23 (d, J = 21.2 Hz), 46.74, 40.59, 37.00,
35.37, 27.30, 10.81. HRMS (ESI) m/z [M + H]+ calcd. for
C26H35F2N2O4 477.2559, found 477.2550.
4.1.3 General procedure to synthesize 9a-b.
To a round-bottomed flask equipped with a magnetic stir bar
was added 8a–b (1.0 eq), 4-fluorophenol (1.2 eq), potassium
phosphate (2.0 eq), and DMA (1.0 M). The reaction vessel was
4.1.5.2 4-(2-Ethylbutyl)-N-(3-(4-fluorophenoxy)-5-(3-fluoroprop-
oxy)phenyl)-4-hydroxypiperidine-1-carboxamide (11b).
o
immersed in a 100 C preheated oil bath for 12 h until the
reaction was completed as determined by TLC. After cooling, the
reaction was diluted with water and extracted with ethyl acetate.
The organic layer was washed with saturated brine and dried over
anhydrous MgSO4. After filtration and concentration, the crude
product was pure enough for the next step.
Compound 11b was eluted with hexane/ethyl acetate (1/1,
V/V) as yellow oil. Yield: 98%, yellow oil. H NMR (400 MHz,
1
CDCl3) δ 7.10 – 6.95 (m, 4H), 6.92 – 6.89 (m, 1H), 6.47 (s, 1H),
6.36 (s, 1H), 6.19 (s, 1H), 4.66 (t, J = 5.8 Hz, 1H), 4.54 (t, J = 5.8
Hz, 1H), 4.04 (t, J = 6.1 Hz, 2H), 3.78 (d, J = 12.8 Hz, 2H), 3.33
– 3.24 (m, 2H), 2.18 – 2.06 (m, 2H), 1.63 – 1.58 (m, 4H), 1.41 –
1.34 (m, 7H), 1.08 (s, 1H), 0.85 (t, J = 6.8 Hz, 6H). 13C NMR
(101 MHz, CDCl3) δ 160.28, 158.93, 158.85 (d, J = 242.4 Hz),
154.47, 152.45 (d, J = 2.0 Hz), 141.37, 120.77 (d, J = 8.1 Hz),
116.21 (d, J = 23.2 Hz), 101.95, 100.60, 99.58, 80.65 (d, J =
164.6 Hz), 70.20, 63.63 (d, J = 6.1 Hz), 46.75, 40.44, 37.05,
35.35, 30.26 (d, J = 20.2 Hz), 27.29, 10.81. HRMS (ESI) m/z [M
+ H]+ calcd. for C27H37F2N2O4 491.2716, found 491.2709.
4.1.3.1 1-(2-Fluoroethoxy)-3-(4-fluorophenoxy)-5-nitrobenzene
(9a).
Yellow oil, yield: 99%. 1H NMR (400 MHz, CDCl3) δ 7.45 (t,
J = 2.1 Hz, 1H), 7.37 (t, J = 2.1 Hz, 1H), 7.14 – 6.99 (m, 4H),
6.83 (t, J = 2.3 Hz, 1H), 4.83 – 4.79 (m, 1H), 4.71 – 4.68 (m,
1H), 4.31 – 4.26 (m, 1H), 4.24 – 4.20 (m, 1H).
4.1.3.2 1-(4-Fluorophenoxy)-3-(3-fluoropropoxy)-5-nitrobenzene
(9b).
Yellow oil, yield: 91%. 1H NMR (400 MHz, CDCl3) δ 7.46 (s,
1H), 7.34 (s, 1H), 7.14 – 6.98 (m, 4H), 6.80 (s, 1H), 4.70 (t, J =
5.9 Hz, 1H), 4.58 (t, J = 5.9 Hz, 1H), 4.19 – 4.11 (m, 2H), 2.25 –
2.14 (m, 2H).
4.1.6 1-(4-Fluorophenoxy)-3-nitro-5-(trifluoromethyl)benzene
(13).
To a round-bottomed flask equipped with a magnetic stir bar
was added 1-fluoro-3-nitro-5-(trifluoromethyl)benzene (2.5 g, 12
mmol), 4-fluorophenol (12) (1.1 g, 10 mmol), potassium
phosphate (4.2 g, 20 mmol), and DMA (15 mL). The reaction
vessel was immersed in a 100 oC preheated oil bath for 12 h until
the reaction was completed as determined by TLC. After cooling,
the reaction 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 crude product was purified on a silica gel
column, eluted with hexane/ethyl acetate (50/1, V/V) to afford
13. Yield: 93%. 1H NMR (400 MHz, CDCl3) δ 8.17 (s, 1H), 7.89
(t, J = 2.1 Hz, 1H), 7.52 (s, 1H), 7.21 – 7.12 (m, 2H), 7.12 – 7.05
(m, 2H).
4.1.4 General procedure to synthesize 10a-b.
To a round-bottomed flask equipped with a magnetic stir bar
were added 9a-b (1.0 eq), 10% Pd/C, and ethyl acetate (0.05 M).
The reaction was bubbled with hydrogen gas for 12 h at room
temperature until the reaction was completed as determined by
TLC. The mixture then was filtered through celite. To the filtrate
was added NaHCO3 (2.0 eq) followed by adding 2,2,2-
trichloroethyl chloroformate (1.0 eq) slowly through syringe
under nitrogen at 0 ºC. The reaction was warmed to room
temperature and stirred for 3 h until the reaction was completed
as determined by TLC. The mixture then was washed with water,
saturated brine and dried over anhydrous MgSO4. After filtration
and concentration, the crude product was used directly for the
next step without further purification.
4.1.7 Synthesis of 1-fluoro-3-(4-fluorophenoxy)-5-nitrobenzene
(14).
To a round-bottomed flask equipped with a magnetic stir bar
were added 1,3-difluoro-5-nitrobenzene (7) (8.0 g, 50 mmol), 4-
fluorophenol (12) (6.2 g, 55 mmol), Cs2CO3 (17.9 g, 55 mmol),
and DMA (80 mL). The reaction vessel was immersed in a 65 oC
preheated oil bath for 12 h until the reaction was completed as
determined by TLC. After cooling, the reaction 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 crude product was
4.1.5 General procedure to synthesize 11a-b.
To a round-bottomed flask equipped with a magnetic stir bar
were added 10a–b (1.0 eq), 6 (1.2 eq), DIPEA (2.0 eq), and
DMA. The reaction vessel was immersed in a 100 oC preheated
oil bath for 12 h until the reaction was completed as determined
by TLC. After cooling, the reaction mixture was diluted with
water and extracted with ethyl acetate. The ethyl acetate layer
was washed with 1 N HCl, saturated brine and dried over
anhydrous MgSO4. After filtration and concentration, the crude
product was purified on a silica gel column, eluted with
hexane/ethyl acetate to afford 11a-b.
1
pure enough for the next step. Yield: 95%. H NMR (400 MHz,
CDCl3) δ 7.61 (d, J = 8.1 Hz, 1H), 7.54 (s, 1H), 7.13 – 7.03 (m,
4H), 6.96 (d, J = 9.2 Hz, 1H).
4.1.8 General procedure to synthesize 15a-b.
To a round-bottomed flask equipped with a magnetic stir bar
was added 14 (1.0 eq), 1-methylpiperazine or 1H-pyrazole (1.0
eq), K2CO3 (1.0 eq), and DMSO (2.0 M). The reaction vessel was
immersed in a 65 oC preheated oil bath for 12 h until the reaction
was completed as determined by TLC. After cooling, the reaction
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 crude
4.1.5.1 4-(2-Ethylbutyl)-N-(3-(2-fluoroethoxy)-5-(4-fluoropheno-
xy)phenyl)-4-hydroxypiperidine-1-carboxamide (11a).
Compound 11a was eluted with hexane/ethyl acetate (2/3,
1
V/V) as yellow oil. Yield: 97%. H NMR (400 MHz, CDCl3) δ
7.08 – 6.74 (m, 5H), 6.57 (s, 1H), 6.51 (s, 1H), 6.21 (s, 1H), 4.78
– 4.72 (m, 1H), 4.67 – 4.60 (m, 1H), 4.22 – 4.16 (m, 1H), 4.15 –
4.09 (m, 1H), 3.83 – 3.72 (m, 2H), 3.35 – 3.22 (m, 2H), 1.64 –
1.57 (m, 4H), 1.42 – 1.31 (m, 8H), 0.85 (t, J = 6.7 Hz, 6H). 13C

product was purified on a silica gel column, eluted with
hexane/ethyl acetate to afford 15a–b.
= 6.8 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 159.17 (d, J =
243.4 Hz), 159.12, 154.56, 152.32 (d, J = 3.0 Hz), 141.87,
141.52, 141.21, 127.21, 121.03 (d, J = 8.8 Hz), 116.54 (d, J =
23.2 Hz), 107.82, 107.19, 105.13, 103.44, 70.31, 46.89, 40.58,
37.18, 35.47, 27.43, 10.96. HRMS (ESI) m/z [M + H]+ calcd. for
C27H34FN4O3 481.2609, found 481.2603.
4.1.8.1 1-(3-(4-Fluorophenoxy)-5-nitrophenyl)-4-methylpiperazi-
ne (15a).
Compound 15a was eluted with hexane/ethyl acetate (1/5,
1
V/V) as yellow oil. Yield: 42%. H NMR (400 MHz, CDCl3) δ
4.1.10.3 4-(2-Ethylbutyl)-N-(3-(4-fluorophenoxy)-5-(trifluorome-
thyl)phenyl)-4-hydroxypiperidine-1-carboxamide (17c).
7.46 (s, 1H), 7.14 – 6.97 (m, 5H), 6.79 (s, 1H), 3.34 – 3.22 (m,
4H), 2.61 – 2.51 (m, 4H), 2.36 (s, 3H).
Compound 17c was eluted with hexane/ethyl acetate (2/1,
1
V/V) as yellow solid. Yield: 44%, M.P. 108 – 111 °C. H NMR
4.1.8.2 1-(3-(4-Fluorophenoxy)-5-nitrophenyl)-1H-pyrazole
(400 MHz, CDCl3) δ 7.23 (d, J = 21.6 Hz, 2H), 7.04 – 6.84 (m,
5H), 6.75 (s, 1H), 3.84 – 3.67 (m, 2H), 3.32 - 3.12 (m, 2H), 1.81
(s, 1H), 1.62 – 1.44 (m, 4H), 1.37 – 1.21 (m, 7H), 0.90 – 0.72 (m,
6H). 13C NMR (101 MHz, CDCl3) δ 159.18 (d, J = 244.4 Hz),
158.54, 154.26, 151.73, 141.51, 132.12 (d, J = 32.3 Hz), 123.55
(d, J = 273.7 Hz), 121.04 (d, J = 8.1 Hz), 116.53 (d, J = 23.2 Hz),
112.04, 110.77, 108.63, 70.14, 46.73, 40.43, 37.01, 35.32, 27.27,
10.78. HRMS (ESI) m/z [M + Na]+ calcd. for C25H30F4N2NaO3
505.2085, found 505.2078.
(15b).
Compound 15b was eluted with hexane/ethyl acetate (5/1,
V/V) as yellow semi-solid. Yield: 53%. H NMR (400 MHz,
CDCl3) δ 8.22 (s, 1H), 7.98 (d, J = 2.4 Hz, 1H), 7.73 (d, J = 12.4
Hz, 2H), 7.62 (s, 1H), 7.18 – 7.05 (m, 4H), 6.53 (s, 1H).
1
4.1.9 General procedure to synthesize 16a-c.
To a round-bottomed flask equipped with a magnetic stir bar
were added 13 or 15a–b (1.0 eq), 10% Pd/C, and ethyl acetate
(0.2 M). The reaction was bubbled with hydrogen gas for 12 h at
room temperature until the reaction was completed as determined
by TLC. The mixture then was filtered through celite. To the
filtrate was added NaHCO3 (2.0 eq) followed by adding 2,2,2-
trichloroethyl chloroformate (1.0 eq) slowly through syringe
under nitrogen at 0 ºC. The reaction was warmed to room
temperature and stirred for 3 h until the reaction was completed
as determined by TLC. Then, the mixture was washed with
water, saturated brine and dried over anhydrous MgSO4. After
filtration and concentration, the crude product was used directly
for the next step without further purification.
4.1.11 General procedure to synthesize 19a-f.
To a round-bottomed flask equipped with a magnetic stir bar
was added 1-fluoro-3-nitro-5-(trifluoromethyl)benzene (18) (1.0
eq), 4-substituted phenol (1.0 eq), potassium phosphate (2 eq),
and DMA (2.0 M). The reaction vessel was immersed in a 100 oC
preheated oil bath for 12 h until the reaction was completed as
determined by TLC. After cooling, the reaction was diluted with
water and extracted with ethyl acetate. The organic layer was
washed with saturated brine and dried over anhydrous MgSO4.
After filtration and concentration, the crude product was purified
on a silica gel column, eluted with hexane/ethyl acetate to afford
19a–f.
4.1.10 General procedure to synthesize 17a-c.
To a round-bottomed flask equipped with a magnetic stir bar
were added 16a–c (1.0 eq), 6 (1.2 eq), DIPEA (2.0 eq) and DMA
(0.5 M). The reaction vessel was immersed in a 100 oC preheated
oil bath for 12 h until the reaction was completed as determined
by TLC. After cooling, the reaction 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 crude product was purified on a
silica gel column to afford 17a–c.
4.1.11.1 1-(4-Methoxyphenoxy)-3-nitro-5-(trifluoromethyl)benze-
ne (19a).
Coupling of 1-fluoro-3-nitro-5-(trifluoromethyl)benzene (18)
(1.0 g, 4.8 mmol) with 4-methoxyphenol (0.5 g, 4.0 mmol)
yielded 19a (1.4 g, 99%), eluted with hexane/ethyl acetate (20/1,
1
V/V). H NMR (400 MHz, CDCl3) δ 8.13 (s, 1H), 7.86 (s, 1H),
7.50 (s, 1H), 7.10 - 6.92 (m, 5H), 3.85 (s, 3H).
4.1.11.2 4-(3-Nitro-5-(trifluoromethyl)phenoxy)benzamide (19b).
Coupling of 1-fluoro-3-nitro-5-(trifluoromethyl)benzene (18)
(1.1 g, 5.0 mmol) with 4-hydroxybenzamide (0.7 g, 5.0 mmol)
yielded 19b (1.6 g, 99%), eluted with hexane/ethyl acetate (1/2,
V/V). H NMR (400 MHz, CDCl3) δ 8.18 (s, 1H), 7.93 (s, 1H),
7.85 (d, J = 8.9 Hz, 2H), 7.53 (s, 1H), 7.08 (d, J = 8.8 Hz, 2H).
4.1.10.1 4-(2-Ethylbutyl)-N-(3-(4-fluorophenoxy)-5-(4-methylpip-
erazin-1-yl)phenyl)-4-hydroxypiperidine-1-carboxamide (17a).
Compound 17a was eluted with ethyl acetate/methanol (5/1,
1
1
V/V) as yellow oil. Yield: 20%, H NMR (400 MHz, CDCl3) δ
7.00 – 6.91 (m, 5H), 6.45 (s, 1H), 6.32 (s, 1H), 6.21 (s, 1H), 3.80
– 3.71 (m, 2H), 3.31 – 3.20 (m, 2H), 3.16 (s, 4H), 2.50 (s, 4H),
2.31 (s, 3H), 1.65 – 1.56 (m, 4H), 1.40 – 1.25 (m, 8H), 0.83 (t, J
= 6.8 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 158.69, 158.68 (d,
J = 242.4), 154.78, 153.02 (d, J = 2.02), 152.93, 141.33, 120.40
(d, J = 8.08), 116.19 (d, J = 23.2), 102.20, 100.93, 100.78, 70.22,
54.99, 48.58, 46.86, 46.11, 40.54, 37.14, 35.42, 27.37, 10.92.
HRMS (ESI) m/z [M + H]+ calcd. for C29H42FN4O3 513.3235,
found 513.3226.
4.1.11.3 N-methyl-4-(3-nitro-5-(trifluoromethyl)phenoxy)benza-
mide (19c).
Coupling of 1-fluoro-3-nitro-5-(trifluoromethyl)benzene (18)
(1.8 g, 8.7 mmol) with 4-hydroxy-N-methylbenzamide (1.1 g, 7.3
mmol) yielded 19c (2.5 g, 99%), eluted with hexane/ethyl acetate
1
(1/2, V/V). H NMR (400 MHz, CDCl3) δ 8.23 (s, 1H), 7.97 (s,
1H), 7.86 (d, J = 8.8 Hz, 2H), 7.58 (s, 1H), 7.13 (d, J = 8.9 Hz,
2H), 6.13 (s, 1H), 3.04 (d, J = 4.9 Hz, 3H).
4.1.10.2 4-(2-Ethylbutyl)-N-(3-(4-fluorophenoxy)-5-(1H-pyrazol-
1-yl)phenyl)-4-hydroxypiperidine-1-carboxamide (17b).
4.1.11.4 N, N-dimethyl-4-(3-nitro-5-(trifluoromethyl)phenoxy)be-
nzamide (19d).
Compound 17b was eluted with hexane/ethyl acetate (2/1,
V/V) as yellow semi-solid. Yield: 30%, H NMR (400 MHz,
CDCl3) δ 7.86 (s, 1H), 7.65 (s, 1H), 7.50 (s, 1H), 7.08 – 6.90 (m,
6H), 6.80 (s, 1H), 6.41 (s, 1H), 3.85 – 3.70 (m, 2H), 3.32 – 3.21
(m, 2H), 1.57 (s, 4H), 1.42 – 1.30 (m, 7H), 1.26 (s, 1H), 0.84 (t, J
1
Coupling of 1-fluoro-3-nitro-5-(trifluoromethyl)benzene (18)
(1.1 g, 5.0 mmol) with 4-hydroxy-N,N-dimethylbenzamide (0.8
g, 5.0 mmol) yielded 19d (1.8 g, 99%), eluted with hexane/ethyl
1
acetate (1/1, V/V). H NMR (400 MHz, CDCl3) δ 8.22 (s, 1H),

7.98 (s, 1H), 7.60 – 7.51 (m, 3H), 7.15 – 7.08 (m, 2H), 3.09 (s,
6H).
4.1.13.2 N-(3-(4-Carbamoylphenoxy)-5-(trifluoromethyl)phenyl)-
4-(2-ethylbutyl)-4-hydroxypiperidine-1-carboxamide (21b).
Coupling of 20b (236 mg, 0.5 mmol) with 6 (95 mg, 0.6
mmol) in the presence of DIPEA afforded 21b (100 mg, 39%),
eluted with ethyl acetate. White solid, M.P. 98 – 101 °C. H
4.1.11.5 Methyl 4-(3-nitro-5-(trifluoromethyl)phenoxy)benzoate
(19e).
1
NMR (400 MHz, DMSO) δ 8.84 (s, 1H), 7.94 (d, J = 8.3 Hz,
3H), 7.76 (s, 1H), 7.49 (s, 1H), 7.34 (s, 1H), 7.11 (d, J = 8.2 Hz,
2H), 6.92 (s, 1H), 3.85 – 3.70 (m, 2H), 3.14 (t, J = 11.6 Hz, 2H),
1.55 – 1.40 (m, 5H), 1.36 – 1.22 (m, 7H), 0.90 (d, J = 6.3 Hz,
6H). 13C NMR (101 MHz, DMSO) δ 167.05, 158.32, 156.82,
154.08, 143.62, 130.52 (d, J = 32.3 Hz), 129.92, 129.83, 123.81
(d, J = 273.7 Hz), 118.38, 111.99, 110.55, 107.92, 68.38, 45.25,
42.06, 38.87, 34.95, 28.75, 11.30. HRMS (ESI) m/z [M + Na]+
calcd. for C26H32F3N3NaO4 530.2243, found 530.2251.
Coupling of 1-fluoro-3-nitro-5-(trifluoromethyl)benzene (18)
(1.1 g, 5.0 mmol) with methyl- 4-hydroxybenzoate (0.8 g, 5.0
mmol) yielded 19e (1.0 g, 60%), eluted with hexane/ethyl acetate
(10/1, V/V). 1H NMR (400 MHz, CDCl3) δ 8.25 (s, 1H), 8.13 (d,
J = 8.2 Hz, 2H), 8.00 (s, 1H), 7.60 (s, 1H), 7.12 (d, J = 8.9 Hz,
2H), 3.94 (s, 3H).
4.1.11.6 1-(4-(Methylsulfonyl)phenoxy)-3-nitro-5-(trifluorometh-
yl)benzene (19f).
Coupling of 1-fluoro-3-nitro-5-(trifluoromethyl)benzene (18)
(2.5 g, 12.0 mmol) with 4-(methylsulfonyl)phenol (1.7 g, 10.0
mmol) yielded 19f (2.8 g, 78%), eluted with hexane/ethyl acetate
4.1.13.3 4-(2-Ethylbutyl)-4-hydroxy-N-(3-(4-(methylcarbamoyl)-
phenoxy)-5 (trifluoromethyl)phenyl)piperidine-1-carboxamide
(21c).
1
(3/2, V/V). H NMR (400 MHz, CDCl3) δ 8.33 – 8.30 (m, 1H),
Coupling of 20c (243 mg, 0.5 mmol) with 6 (112 mg, 0.6
mmol) in the presence of DIPEA afforded 21c (117 mg, 45%),
eluted with hexane/ethyl acetate (1/7, V/V). White solid, M.P. 98
– 100 °C. 1H NMR (400 MHz, DMSO) δ 8.84 (s, 1H), 8.41 (d, J
= 4.4 Hz, 1H), 7.89 (d, J = 8.6 Hz, 2H), 7.76 (s, 1H), 7.50 (s,
1H), 7.12 (d, J = 8.6 Hz, 2H), 6.91 (s, 1H), 3.79 (d, J = 13.1 Hz,
2H), 3.13 (t, J = 11.4 Hz, 2H), 2.78 (d, J = 4.4 Hz, 3H), 1.52 –
1.33 (m, 5H), 1.32 – 1.21 (m, 7H), 0.80 (t, J = 7.3 Hz, 6H). 13C
NMR (101 MHz, DMSO) δ 166.22, 158.58, 157.24, 154.48,
144.05, 130.94 (d, J = 31.3 Hz), 130.58, 129.77, 124.24 (d, J =
270.7 Hz), 118.85, 112.44, 110.97, 108.35, 68.87, 46.53, 37.13,
35.06, 31.09, 27.18, 26.67, 11.15. HRMS (ESI) m/z [M + Na]+
calcd. for C27H34F3N3NaO4 544.2394, found 544.2391.
8.07 – 8.01 (m, 3H), 7.67 – 7.63 (m, 1H), 7.26 – 7.20 (m, 2H),
3.11 (s, 3H).
4.1.12 General procedure to synthesize 20a-f.
To a round-bottomed flask equipped with a magnetic stir bar
were added 19a-f (1.0 eq), 10% Pd/C and ethyl acetate (0.2 M).
The reaction was bubbled with hydrogen gas for 12 h at room
temperature until the reaction was completed as determined by
TLC. The mixture then was filtered through celite. To the filtrate
was added NaHCO3 (2.0 eq) followed by adding 2,2,2-
trichloroethyl chloroformate (1.0 eq) slowly through syringe
under nitrogen at 0 ºC. The reaction was warmed to room
temperature and stirred for 3 h until the reaction was completed
as determined by TLC. Then, the mixture was washed with
water, saturated brine and dried over anhydrous MgSO4. After
filtration and concentration, the crude product was used directly
for the next step without further purification.
4.1.13.4 N-(3-(4-(Dimethylcarbamoyl)phenoxy)-5-(trifluorometh-
yl)phenyl)-4-(2-ethylbutyl)-4-hydroxypiperidine-1-carboxamide
(21d).
Coupling of 20d (250 mg, 0.5 mmol) with 6 (95 mg, 0.6
mmol) in the presence of DIPEA afforded 21d (180 mg, 67%),
eluted with hexane/ethyl acetate (1/10, V/V). White solid, M.P.
82 – 84 °C. 1H NMR (400 MHz, CDCl3) δ 7.82 (s, 1H), 7.56 (s,
1H), 7.33 (d, J = 6.0 Hz, 2H), 7.13 (s, 1H), 6.97 (d, J = 6.1 Hz,
2H), 6.89 (s, 1H), 3.90 – 3.65 (m, 2H), 3.30 – 2.86 (m, 8H), 1.56
– 1.44 (m, 6H), 1.40 – 1.30 (m, 5H), 1.25 (s, 1H), 0.93 (d, J = 5.6
Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 171.43, 157.69, 157.31,
154.67, 142.48, 132.35 (d, J = 33.3 Hz), 131.41, 129.12, 123.80
(d, J = 273.7 Hz), 118.95, 112.28, 111.52, 109.57, 70.14, 45.07,
42.54, 37.27, 35.01, 34.62, 28.38, 11.62. HRMS (ESI) m/z [M +
Na]+ calcd. for C28H36F3N3NaO4 558.2556, found 558.2661.
4.1.13 General procedure to synthesize 21a-f.
To a round-bottomed flask equipped with a magnetic stir bar
were added 20a-f (1.0 eq), 6 (1.2 eq), DIPEA (2.0 eq), and DMA
(1.0 M). The reaction vessel was immersed in a 100 oC preheated
oil bath for 12 h until the reaction was completed as determined
by TLC. After cooling, the reaction 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 crude product was purified on a
silica gel column to afford 21a–f.
4.1.13.1 4-(2-Ethylbutyl)-4-hydroxy-N-(3-(4-methoxyphenoxy)-5-
(trifluoromethyl)phenyl)piperidine-1-carboxamide (21a).
4.1.13.5 Methyl 4-(3-(4-(2-ethylbutyl)-4-hydroxypiperidine-1-
carboxamido)-5-(trifluoromethyl)phenoxy)benzoate (21e).
Coupling of 20a (229 mg, 0.5 mmol) with 6 (86 mg, 0.6
mmol) in the presence of DIPEA afforded 21a (200 mg, 81%),
eluted with hexane/ethyl acetate (1/1, V/V). White solid, M.P. 79
– 81 °C. 1H NMR (400 MHz, CDCl3) δ 7.36 (s, 1H), 7.15 (s, 1H),
6.99 (d, J = 9.0 Hz, 2H), 6.90 (d, J = 9.0 Hz, 2H), 6.82 (s, 1H),
6.46 (s, 1H), 3.90 – 3.83 (m, 2H), 3.81 (s, 3H), 3.32 – 3.22 (m,
2H), 1.65 – 1.58 (m, 5H), 1.35 – 1.18 (m, 6H), 1.04 (s, 1H), 0.93
(d, J = 6.9 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 159.35,
156.33, 154.18, 149.02, 141.31, 132.07 (d, J = 33.3 Hz), 123.60
(d, J = 266.6 Hz), 121.13, 115.01, 111.32, 110.19, 108.23, 71.48,
55.57, 45.60, 40.34, 38.02, 33.75, 28.06, 11.34. HRMS (ESI) m/z
[M + Na]+ calcd. for C26H33F3N2NaO4 517.2290, found 517.2299.
Coupling of 20e (487 mg, 1.0 mmol) with 6 (189 mg, 1.2
mmol) in the presence of DIPEA afforded 21e (280 mg, 54%),
eluted with hexane/ethyl acetate (1/1, V/V). White solid, M.P. 80
– 82 °C. 1H NMR (400 MHz, CDCl3) δ 7.68 (d, J = 8.3 Hz, 2H),
7.42 (s, 1H), 7.00 (d, J = 8.3 Hz, 2H), 6.93 (s, 2H), 6.31 (s, 1H),
3.93 – 3.83 (m, 2H), 3.30 – 3.18 (m, 2H), 2.98 (s, 3H), 1.60 –
1.54 (m, 5H), 1.30 – 1.24 (m, 6H), 1.10 (s, 1H), 0.92 (d, J = 6.8
Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 166.51, 160.64, 156.63,
154.08, 141.72, 132.49 (d, J = 33.3 Hz), 131.79, 125.27, 123.44
(d, J = 272.7 Hz), 117.92, 113.65, 111.88, 110.46, 70.13, 52.07,
45.97, 42.47, 37.10, 34.83, 27.28, 11.02. HRMS (ESI) m/z [M +
Na]+ calcd. for C27H33F3N2NaO5 545.2239, found 545.2230.

4.1.13.6 4-(2-Ethylbutyl)-4-hydroxy-N-(3-(4-(methylsulfonyl)phe-
4.1.14.6 3-Isobutylpyrrolidin-3-ol (24b).
noxy)-5-(trifluoromethyl)phenyl)piperidine-1-carboxamide (21f).
1
Yield: 92%. H NMR (400 MHz, CDCl3) δ 3.75 – 3.41 (m,
Coupling of 20f (253 mg, 0.5 mmol) with 6e (112 mg, 0.6
mmol) in the presence of DIPEA afforded 21f (160 mg, 59%),
eluted with hexane/ethyl acetate (1/2, V/V). White solid, M.P. 86
– 88 °C. 1H NMR (400 MHz, CDCl3) δ 7.79 (d, J = 7.9 Hz, 2H),
7.47 – 7.36 (m, 2H), 7.22 – 7.16 (m, 1H), 7.01 (d, J = 7.9 Hz,
2H), 6.85 (s, 1H), 3.83 – 3.65 (m, 2H), 3.25 – 3.10 (m, 2H), 2.98
(s, 3H), 1.56 – 1.37 (m, 5H), 1.36 – 1.19 (m, 7H), 0.76 (t, J = 6.9
Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 161.52, 155.62, 154.22,
142.24, 134.54, 132.48 (d, J = 32.3 Hz), 129.73, 123.39 (d, J =
273.7 Hz), 118.23, 114.22, 112.59, 110.56, 70.07, 46.71, 44.71,
40.42, 37.00, 35.29, 27.25, 10.79. HRMS (ESI) m/z [M + Na]+
calcd. for C26H33F3N2NaO5S 565.1954, found 565.1944.
4H), 2.12 – 1.78 (m, 4H), 1.71 – 1.49 (m, 3H), 1.02 – 0.91 (m,
6H).
4.1.15 General procedure to synthesize 25a-j.
To a round-bottomed flask equipped with a magnetic stir bar
were added 20c or 20f (1.0 eq), 22a-d or 24a-b (1.2 eq), DIPEA
(2.0 eq), and DMA (1.0 M). The reaction vessel was immersed in
o
a 100 C preheated oil bath for 12 h until the reaction was
completed as determined by TLC. After cooling, the reaction 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 crude
product was purified on a silica gel column to afford 25a–j.
4.1.14 General procedure to synthesize 22a-d and 24a-b.
To a dried round two neck bottomed flask equipped with a
magnetic stir bar were added 0.6 M LaCl3-2LiCl in THF (1.5 eq)
under nitrogen, Grignard reagents in THF (1.5 eq) was added
slowly through syringe at 0 ºC. After stirring at room temperature
for 3 h, a solution of ketones (5 or 23a-b) (1.0 eq) in THF (1.0
M) was added into the mixture. The reaction was stirred for
another18 h until the reaction was completed as determined by
TLC and then quenched with 25% acetic acid. The mixture was
extracted with ethyl acetate, the ethyl acetate layer was washed
with saturated brine and dried over anhydrous MgSO4. After
filtration and concentration, the crude product was used directly
for the next step without purification.
To a round-bottomed flask equipped with a magnetic stir bar
were added above crude product, 10% Pd/C and methanol (0.5
M). The reaction was bubbled with hydrogen gas for 6 h at room
temperature until the reaction was completed as determined by
TLC and then filtered through celite. The filtrate was
concentrated under reduced pressure to afford yellow oil product
22a-d and 24a-b.
4.1.15.1 4-Hydroxy-4-methyl-N-(3-(4-(methylcarbamoyl)phenox-
y)-5-(trifluoromethyl)phenyl)piperidine-1-carboxamide (25a).
Coupling of 20c (243 mg, 0.5 mmol) with 22a (69 mg, 0.6
mmol) in the presence of DIPEA afforded 25a (160 mg, 62%),
eluted with hexane/ethyl acetate (1/5, V/V). White solid, M.P.
103 – 106 °C. 1H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 8.8 Hz,
2H), 7.50 (s, 1H), 7.40 (s, 1H), 7.27 (s, 1H), 7.12 (d, J = 8.7 Hz,
2H), 6.98 (s, 1H), 6.61 (s, 1H), 3.79 – 3.72 (m, 2H), 3.42 – 3.32
(m, 2H), 3.06 (s, 3H), 1.67 – 1.61 (m, 4H), 1.31 (s, 3H). 13C
NMR (101 MHz, DMSO) δ 165.80, 158.15, 156.81, 154.07,
143.60, 130.51 (d, J = 32.3 Hz), 130.16, 129.33, 123.79 (d, J =
273.7 Hz), 118.42, 112.05, 110.59, 107.92, 66.02, 40.34, 38.21,
29.70, 26.23. HRMS (ESI) m/z [M
C22H25F3N3O4 452.1792, found 452.1787.
+
Na]+ calcd. for
4.1.15.2 4-Hydroxy-4-methyl-N-(3-(4-(methylsulfonyl)phenoxy)-
5-(trifluoromethyl)phenyl)piperidine-1-carboxamide (25b).
Coupling of 20f (253 mg, 0.5 mmol) with 22a (69 mg, 0.6
mmol) in the presence of DIPEA afforded 25b (160 mg, 68%),
eluted with hexane/ethyl acetate (1/2, V/V). White solid, M.P. 94
– 96 °C. 1H NMR (400 MHz, CDCl3) δ 7.68 (d, J = 7.4 Hz, 2H),
7.42 (s, 1H), 7.03 – 6.91 (m, 4H), 6.32 (s, 1H), 3.84 – 3.69 (m,
2H), 3.41 – 3.25 (m, 2H), 2.98 (s, 3H), 1.63 – 1.56 (m, 5H), 1.28
(s, 3H). 13C NMR (101 MHz, CDCl3) δ 161.50, 155.71, 154.15,
142.11, 134.66, 132.59 (d, J = 32.3 Hz), 129.74, 123.37 (d, J =
273.7 Hz), 118.26, 114.14, 112.54, 110.71, 67.66, 44.67, 40.71,
4.1.14.1 4-Methylpiperidin-4-ol (22a).
1
Yield: 93%. H NMR (400 MHz, CDCl3) δ 5.86 (s, 1H), 3.03
– 2.87 (m, 2H), 2.48 – 2.34 (m, 2H), 1.68 – 1.55 (m, 4H), 1.26 (s,
3H).
4.1.14.2 4-Ethylpiperidin-4-ol (22b).
Yield: 98%. H NMR (400 MHz, CDCl3) δ 5.82 (s, 1H), 3.00
38.25, 30.18. HRMS (ESI) m/z [M
C21H24F3N2O5S 473.1353, found 473.1343.
+
H]+ calcd. for
1
– 2.86 (m, 2H), 2.44 – 2.23 (m, 2H), 1.72 – 1.39 (m, 7H), 0.95 –
0.80 (m, 3H).
4.1.15.3 4-Ethyl-4-hydroxy-N-(3-(4-(methylcarbamoyl)phenoxy)-
5-(trifluoromethyl)phenyl)piperidine-1-carboxamide (25c).
4.1.14.3 4-Isopropylpiperidin-4-ol (22c).
1
Coupling of 20c (243 mg, 0.5 mmol) with 22b (78 mg, 0.6
mmol) in the presence of DIPEA afforded 25c (160 mg, 69%),
eluted with ethyl acetate. White solid, M.P. 104 – 107 °C. H
Yield: 97%. H NMR (400 MHz, CDCl3) δ 3.06 – 2.84 (m,
4H), 1.67 – 1.51 (m, 5H), 0.90 (d, J = 6.9 Hz, 6H).
1
NMR (400 MHz, CDCl3) δ 7.70 (d, J = 8.5 Hz, 2H), 7.42 (s, 1H),
7.28 (s, 1H), 7.01 (d, J = 8.4 Hz, 2H), 6.94 (s, 1H), 6.84 (s, 1H),
6.24 (s, 1H), 3.86 – 3.76 (m, 2H), 3.35 – 3.24 (m, 2H), 2.99 (d, J
= 4.8 Hz, 3H), 1.61 – 1.53 (m, 6H), 1.12 (s, 1H), 0.93 (t, J = 7.5
Hz, 3H). 13C NMR (101 MHz, DMSO) δ 165.79, 158.14, 156.81,
154.06, 143.61, 130.50 (d, J = 32.3 Hz), 130.16, 129.33, 123.79
(d, J = 273.7 Hz), 118.42, 112.04, 110.61, 107.89, 67.83, 40.05,
35.85, 34.78, 26.22, 7.14. HRMS (ESI) m/z [M + H]+ calcd. for
C23H27F3N3O4 466.1948, found 466.1940.
4.1.14.4 4-Isobutylpiperidin-4-ol (22d).
1
Yield: 95%. H NMR (400 MHz, CDCl3) δ 3.44 (s, 1H), 3.38
– 3.17 (m, 4H), 1.88 – 1.76 (m, 4H), 1.48 – 1.40 (m, 2H), 1.24 –
1.17 (m, 2H), 0.95 (d, J = 6.1 Hz, 6H).
4.1.14.5 3-Isobutylazetidin-3-ol (24a).
1
Yield: 93%. H NMR (400 MHz, CDCl3) δ 3.95 – 3.87 (m,
2H), 3.75 – 3.65 (m, 2H), 1.96 – 1.82 (m, 1H), 1.75 – 1.58 (m,
2H), 0.91 (d, J = 5.8 Hz, 6H).
4.1.15.4 4-Ethyl-4-hydroxy-N-(3-(4-(methylsulfonyl)phenoxy)-5-
(trifluoromethyl)phenyl)piperidine-1-carboxamide (25d).
Coupling of 20f (253 mg, 0.5 mmol) with 22b (78 mg, 0.6
mmol) in the presence of DIPEA afforded 25d (240 mg, 99%),

eluted with hexane/ethyl acetate (1/2, V/V). White solid, M.P. 89
– 91 °C. 1H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 8.7 Hz, 2H),
7.50 (s, 1H), 7.41 (s, 1H), 7.12 (d, J = 8.7 Hz, 2H), 6.97 (s, 1H),
6.66 (s, 1H), 3.86 – 3.75 (m, 2H), 3.38 – 3.27 (m, 2H), 3.06 (s,
3H), 1.65 – 1.54 (m, 6H), 1.11 (s, 1H), 0.94 (t, J = 7.5 Hz, 3H).
13C NMR (101 MHz, CDCl3) δ 161.51, 155.68, 154.16, 142.17,
134.64, 132.56 (d, J = 33.3 Hz), 129.73, 123.38 (d, J = 273.7
Hz), 118.23, 114.15, 112.56, 110.68, 110.67, 44.67, 40.45, 36.04,
(m, 2H), 3.41 – 3.24 (m, 2H), 3.06 (s, 3H), 1.94 – 1.70 (m, 1H),
1.71 – 1.40 (m, 7H), 1.07 – 0.90 (m, 6H). 13C NMR (101 MHz,
CDCl3) δ 161.52, 155.64, 154.20, 142.21, 134.58, 132.51 (d, J =
32.3 Hz), 129.73, 123.38 (d, J = 275.7 Hz), 118.22, 114.17,
112.55, 110.60, 70.03, 51.89, 44.67, 40.42, 37.05, 24.82, 23.24.
HRMS (ESI) m/z [M + Na]+ calcd. for C24H29F3N2NaO5S
537.1641, found 537.1641.
35.52, 6.96. HRMS (ESI) m/z [M
C22H26F3N2O5S 487.1509, found 487.1502.
+
H]+ calcd. for
4.1.15.9 3-Hydroxy-3-isobutyl-N-(3-(4-(methylcarbamoyl)pheno-
xy)-5-(trifluoromethyl)phenyl)azetidine-1-carboxamide (25i).
Coupling of 20c (243 mg, 0.5 mmol) with 24a (78 mg, 0.6
mmol) in the presence of DIPEA afforded 25i (110 mg, 47%),
eluted with hexane/ethyl acetate (1/5, V/V). White solid, M.P.
4.1.15.5 4-Hydroxy-4-isopropyl-N-(3-(4-(methylcarbamoyl)phen-
oxy)-5-(trifluoromethyl)phenyl)piperidine-1-carboxamide (25e).
1
Coupling of 20c (243 mg, 0.5 mmol) with 22c (86 mg, 0.6
mmol) in the presence of DIPEA afforded 25e (108 mg, 44%),
107 – 109 °C. H NMR (400 MHz, DMSO) δ 8.80 (s, 1H), 8.42
(s, 1H), 7.90 (d, J = 7.4 Hz, 2H), 7.77 (s, 1H), 7.52 (s, 1H), 7.12
(d, J = 7.5 Hz, 2H), 6.92 (s, 1H), 3.95 – 3.65 (m, 4H), 2.78 (s,
3H), 1.91 – 1.77 (m, 1H), 1.65 – 1.45 (m, 2H), 1.22 (s, 1H), 0.88
(d, J = 5.3 Hz, 6H). 13C NMR (101 MHz, DMSO) δ 166.22,
158.46, 157.45, 156.37, 143.47, 131.11 (d, J = 32.3 Hz), 130.68,
129.79, 124.19 (d, J = 273.7 Hz), 118.98, 111.63, 110.24, 108.36,
69.11, 63.86, 47.30, 26.67, 24.52, 23.50. HRMS (ESI) m/z [M +
H]+ calcd. for C23H27F3N3O4 466.1948, found 466.1939.
1
eluted with ethyl acetate. White solid, M.P. 100 – 102 °C. H
NMR (400 MHz, CDCl3) δ 8.03 (d, J = 8.5 Hz, 2H), 7.40 (d, J =
11.8 Hz, 2H), 7.26 (s, 1H), 7.02 (d, J = 8.5 Hz, 2H), 6.95 (s, 1H),
6.52 (s, 1H), 3.91 (s, 3H), 3.83 – 3.76 (m, 2H), 3.38 – 3.28 (m,
2H), 1.90 – 1.80 (m, 1H), 1.65 – 1.62 (m, 2H), 1.45 – 1.41 (m,
2H), 1.06 (s, 1H), 0.98 (d, J = 6.6 Hz, 6H). 13C NMR (101 MHz,
CDCl3) δ 168.00, 158.89, 157.12, 154.51, 141.94, 132.28 (d, J =
33.3 Hz), 130.07, 128.89, 123.53 (d, J = 374.7 Hz), 118.55,
112.60, 111.58, 109.88, 71.42, 40.25, 38.00, 33.77, 26.75, 16.33.
HRMS (ESI) m/z [M + H]+ calcd. for C24H29F3N3O4 480.2105,
found 480.2097.
4.1.15.10 3-Hydroxy-3-isobutyl-N-(3-(4-(methylcarbamoyl)phen-
oxy)-5-(trifluoromethyl)phenyl)pyrrolidine-1-carboxamide (25j).
Coupling of 20c (243 mg, 0.5 mmol) with 24b (86 mg, 0.6
mmol) in the presence of DIPEA afforded 25j (100 mg, 42%),
eluted with hexane/ethyl acetate (1/5, V/V). White solid, M.P.
4.1.15.6 4-Hydroxy-4-isopropyl-N-(3-(4-(methylsulfonyl)phenox-
y)-5-(trifluoromethyl)phenyl)piperidine-1-carboxamide (25f).
1
113 – 116 °C. H NMR (400 MHz, DMSO) δ 8.48 (d, J = 45.8
Coupling of 20f (253 mg, 0.5 mmol) with 22c (86 mg, 0.6
mmol) in the presence of DIPEA afforded 25f (200 mg, 80%),
eluted with hexane/ethyl acetate (1/2, V/V). White solid, M.P. 92
– 104 °C. 1H NMR (400 MHz, CDCl3) δ 7.91 (d, J = 8.8 Hz, 2H),
7.50 (s, 1H), 7.41 (s, 1H), 7.12 (d, J = 8.8 Hz, 2H), 6.97 (s, 1H),
6.63 (s, 1H), 3.91 – 3.84 (m, 2H), 3.32 – 3.23 (m, 2H), 3.06 (s,
3H), 1.66 – 1.58 (m, 5H), 1.06 (s, 1H), 0.93 (d, J = 6.9 Hz, 6H).
13C NMR (101 MHz, CDCl3) δ 161.50, 155.71, 154.08, 142.14,
134.68, 132.59 (d, J = 33.3 Hz), 129.74, 123.38 (d, J = 271.7
Hz), 118.23, 114.12, 112.48, 110.69, 71.43, 44.67, 40.34, 38.02,
Hz, 2H), 8.11 – 7.43 (m, 4H), 7.13 (s, 2H), 6.93 (s, 1H), 3.55 –
3.40 (m, 2H), 2.90 – 2.67 (m, 2H), 2.10 – 1.68 (m, 3H), 1.60 –
1.37 (m, 2H), 1.24 (s, 1H), 0.93 (s, 6H). 13C NMR (101 MHz,
DMSO) δ 165.81, 158.17, 156.81, 153.43, 143.45, 130.52 (d, J =
32.3 Hz), 130.15, 129.35, 123.82 (d, J = 271.7 Hz), 118.41,
111.94, 110.49, 107.89, 58.16, 47.16, 45.37, 44.48, 26.25, 24.37,
24.18, 24.13. HRMS (ESI) m/z [M + H]+ calcd. for C24H29F3N3O4
480.2105, found 480.2098.
4.1.16 General procedure to synthesize 26a-e.
33.75, 16.36. HRMS (ESI) m/z [M
C23H28F3N2O5S 501.1659, found 501.1666.
+
H]+ calcd. for
To a round-bottomed flask equipped with a magnetic stir bar
was added 14 (1.0 eq), corresponding phenols (1.0 eq), K3PO4
(2.0 eq) and DMA (1.0 M). The reaction vessel was immersed in
4.1.15.7 4-Hydroxy-4-isobutyl-N-(3-(4-(methylcarbamoyl)pheno-
xy)-5-(trifluoromethyl)phenyl)piperidine-1-carboxamide (25g).
o
a 100 C preheated oil bath for 12 h until the reaction was
completed as determined by TLC. After cooling, the reaction was
diluted with water and extracted with ethyl acetate. The organic
layer was washed with saturated brine and dried over anhydrous
MgSO4. After filtration and concentration, the crude product was
purified on a silica gel column, eluted with hexane/ethyl acetate
to afford 26a–e.
Coupling of 20c (243 mg, 0.5 mmol) with 22d (94 mg, 0.6
mmol) in the presence of DIPEA afforded 25g (159 mg, 64%),
eluted with hexane/ethyl acetate (1/7, V/V). White solid, M.P.
190 – 192 °C. 1H NMR (400 MHz, DMSO) δ 8.80 (s, 1H), 8.38
(s, 1H), 7.86 (d, J = 7.5 Hz, 2H), 7.72 (s, 1H), 7.47 (s, 1H), 7.09
(d, J = 7.4 Hz, 2H), 6.88 (s, 1H), 3.85 – 3.65 (m, 2H), 3.20 – 3.00
(m, 2H), 2.75 (s, 3H), 2.47 (s, 1H), 1.85 – 1.70 (m, 1H), 1.52 –
1.21 (m, 6H), 0.87 (d, J = 5.5 Hz, 6H). 13C NMR (101 MHz,
DMSO) δ 166.21, 158.57, 157.24, 154.50, 147.82, 144.04,
140.77, 131.09, 130.58, 129.77, 118.86, 112.45, 109.69 (d, J =
267.7 Hz), 68.79, 51.67, 40.48, 37.29, 26.67, 25.37, 23.17.
HRMS (ESI) m/z [M + Na]+ calcd. for C25H30F3N3NaO4
516.2081, found 516.2069.
4.1.16.1 1-(4-Fluorophenoxy)-3-(4-methoxyphenoxy)-5-nitroben-
zene (26a).
Compound 26a was eluted with hexane/ethyl acetate (10/1,
1
V/V). Yield: 93%. H NMR (400 MHz, CDCl3) δ 7.34 (dd, J =
4.8, 2.1 Hz, 2H), 7.12 – 6.96 (m, 6H), 6.96 – 6.89 (m, 2H), 6.88 –
6.84 (m, 1H), 3.80 (s, 3H).
4.1.16.2 6-(3-(4-Fluorophenoxy)-5-nitrophenoxy)-2-methyl-3,4-
dihydroisoquinolin-1(2H)-one (26b).
4.1.15.8 4-Hydroxy-4-isobutyl-N-(3-(4-(methylsulfonyl)phenoxy)-
5-(trifluoromethyl)phenyl)piperidine-1-carboxamide (25h).
Compound 26b was pure enough for next step. Yield: 99%.
1H NMR (400 MHz, CDCl3) δ 8.12 (d, J = 8.6 Hz, 1H), 7.49 (d, J
= 12.3 Hz, 1H), 7.24 (s, 1H), 7.17 – 6.90 (m, 6H), 6.83 (s, 1H),
3.59 (t, J = 6.6 Hz, 2H), 3.16 (s, 3H), 3.00 (t, J = 6.6 Hz, 2H).
Coupling of 20f (253 mg, 0.5 mmol) with 22d (94 mg, 0.6
mmol) in the presence of DIPEA afforded 25h (210 mg, 82%),
eluted with hexane/ethyl acetate (1/2, V/V). White solid, M.P. 87
– 90 °C. 1H NMR (400 MHz, CDCl3) δ 7.90 (s, 2H), 7.50 (s, 1H),
7.42 (s, 1H), 7.12 (s, 2H), 6.97 (s, 1H), 6.72 (s, 1H), 3.87 – 3.72

4.1.16.3 6-(3-(4-Fluorophenoxy)-5-nitrophenoxy)-3,4-dihydrois-
oquinolin-1(2H)-one (26c).
Compound 26c was pure enough for next step. Yield: 40%. 1H
NMR (400 MHz, CDCl3) δ 8.11 (s, 1H), 7.74 – 7.35 (m, 2H),
7.19 – 6.78 (m, 6H), 6.65 (s, 1H), 3.74 – 3.47 (m, 2H), 3.11 –
2.84 (m, 2H).
1H), 6.39 (t, J = 2.1 Hz, 1H), 4.77 (s, 2H), 4.66 (t, J = 4.7 Hz,
1H), 4.54 (t, J = 4.7 Hz, 1H), 3.81 (dd, J = 10.3, 5.0 Hz, 1H),
3.74 (dd, J = 10.2, 5.1 Hz, 1H).
4.1.17.5 2,2,2-Trichloroethyl (3-(4-fluorophenoxy)-5-((4-(methyl-
sulfonyl)phenyl)thio)phenyl)carbamate (27e).
1
Yield: 99%. H NMR (400 MHz, CDCl3) δ 7.81 (d, J = 8.5
4.1.16.4 N-(2-Fluoroethyl)-4-(3-(4-fluorophenoxy)-5-nitropheno-
Hz, 2H), 7.36 (d, J = 8.6 Hz, 2H), 7.12 (s, 1H), 7.08 – 6.98 (m,
xy)benzamide (26d).
6H), 6.90 (s, 1H), 4.79 (s, 2H), 3.04 (s, 3H).
Compound 26d was eluted with hexane/ethyl acetate (3/2,
V/V). Yield: 31%. 1H NMR (400 MHz, CDCl3) δ 7.85 (d, J = 8.7
Hz, 2H), 7.51 – 7.45 (m, 2H), 7.14 – 7.10 (m, 4H), 7.08 – 7.04
(m, 2H), 6.95 (t, J = 2.2 Hz, 1H), 6.48 (s, 1H), 4.68 (t, J = 4.7 Hz,
1H), 4.56 (t, J = 4.7 Hz, 1H), 3.83 (dd, J = 10.2, 5.1 Hz, 1H),
3.76 (dd, J = 10.2, 5.1 Hz, 1H).
4.1.18 General procedure to synthesize 28a-g.
To a round-bottomed flask equipped with a magnetic stir bar
were added 27a-e (1.0 eq), 6 or 22d (1.2 eq), DIPEA (2.0 eq) and
DMA. The reaction vessel was immersed in a 100 oC preheated
oil bath for 12 h until the reaction was completed as determined
by TLC. After cooling, the reaction 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 crude product was purified on a
silica gel column, eluted with hexane/ethyl acetate to afford 28a–
g.
4.1.16.5 (3-(4-Fluorophenoxy)-5-nitrophenyl)(4-(methylsulfonyl-
)phenyl)sulfane (26e).
Compound 26e was eluted with hexane/ethyl acetate (2/1,
V/V). Yield: 15%. 1H NMR (400 MHz, CDCl3) δ 7.92 – 7.88 (m,
3H), 7.65 (s, 1H), 7.47 (d, J = 8.3 Hz, 2H), 7.28 (s, 1H), 7.16 –
7.02 (m, 5H), 3.08 (s, 3H).
4.1.18.1 4-(2-Ethylbutyl)-N-(3-(4-fluorophenoxy)-5-(4-methoxyp-
henoxy)phenyl)-4-hydroxypiperidine-1-carboxamide (28a).
4.1.17 General procedure to synthesize 27a-e.
Compound 28a was eluted with hexane/ethyl acetate (1/1,
V/V) as white solid. Yield: 52%, M.P. 79 – 80 °C. 1H NMR (400
MHz, CDCl3) δ 6.98 (tt, J = 10.2, 3.0 Hz, 6H), 6.88 – 6.83 (m,
2H), 6.75 (t, J = 2.0 Hz, 1H), 6.64 (t, J = 2.0 Hz, 1H), 6.57 (s,
1H), 6.25 (t, J = 2.2 Hz, 1H), 3.79 (s, 3H), 3.72 (d, J = 12.9 Hz,
2H), 3.31 – 3.21 (m, 2H), 1.59 (dd, J = 8.8, 3.8 Hz, 4H), 1.36
(dd, J = 15.0, 8.8 Hz, 7H), 0.84 (t, J = 7.2 Hz, 6H). 13C NMR
(101 MHz, CDCl3) δ 160.07, 159.12, 158.91 (d, J = 243.4),
156.09, 154.05, 152.28 (d, J = 3.0), 149.38, 141.13, 121.07,
120.76 (d, J = 8.1), 116.26 (d, J = 23.2), 114.84, 103.44, 103.22,
102.33, 70.12, 55.60, 46.71, 40.74, 36.90, 35.36, 27.30, 10.82.
HRMS (ESI) m/z [M + H]+ calcd. for C31H38FN2O5 537.2759,
found 537.2751.
To a round-bottomed flask equipped with a magnetic stir bar
were added 26a–e (1.0 eq), 10% Pd/C and ethyl acetate. The
reaction was bubbled with hydrogen gas for 16 h at room
temperature until the reaction was completed as determined by
TLC. The mixture then was filtered through celite. To the filtrate
was added NaHCO3 (2.0 eq) followed by adding 2,2,2-
trichloroethyl chloroformate (1.0 eq) slowly through syringe
under nitrogen at 0 ºC. The reaction was warmed to room
temperature and stirred for 3 h until the reaction was completed
as determined by TLC. Then, the mixture was washed with
water, saturated brine and dried over anhydrous MgSO4. After
filtration and concentration, the crude product was directly used
for next step without purification.
4.1.18.2 4-(2-Ethylbutyl)-N-(3-(4-fluorophenoxy)-5-((2-methyl-1-
oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)oxy)phenyl)-4-hydroxypi-
peridine-1-carboxamide (28b).
4.1.17.1 2,2,2-Trichloroethyl (3-(4-fluorophenoxy)-5-(4-methoxy-
phenoxy)phenyl)carbamate (27a).
1
Yield: 96%. H NMR (400 MHz, CDCl3) δ 7.05 – 6.97 (m,
Compound 28b was eluted with hexane/ethyl acetate (1/3,
V/V) as white solid. Yield: 24%, M.P. 80 – 82 °C. 1H NMR (400
MHz, CDCl3) δ 8.00 (d, J = 8.4 Hz, 1H), 7.07 – 6.95 (m, 3H),
6.94 – 6.84 (m, 2H), 6.81 (s, 1H), 6.75 (s, 1H), 6.57 (s, 1H), 6.32
(s, 1H), 3.85 – 3.71 (m, 2H), 3.53 (t, J = 6.5 Hz, 2H), 3.34 – 3.20
(m, 2H), 3.12 (s, 3H), 2.94 (t, J = 6.5 Hz, 2H), 1.62 – 1.52 (m,
4H), 1.42 – 1.30 (m, 7H), 1.16 (s, 1H), 0.84 (t, J = 6.6 Hz, 6H).
13C NMR (101 MHz, CDCl3) δ 164.49, 159.90, 159.14, 158.92
(d, J = 243.4 Hz), 157.12, 154.47, 152.17 (d, J = 2.0 Hz), 142.20,
140.19, 130.13, 124.12, 120.82 (d, J = 8.1 Hz), 116.48, 116.28
(d, J = 23.2 Hz), 116.00, 105.62, 105.11, 103.78, 70.06, 47.98,
46.73, 40.38, 37.05, 35.28, 35.04, 27.91, 27.26, 10.80. HRMS
(ESI) m/z [M + Na]+ calcd. for C34H40FN3NaO5 612.2844, found
612.2846.
6H), 6.90 – 6.87 (m, 2H), 6.79 (s, 1H), 6.76 (s, 1H), 6.67 (s, 1H),
6.32 (t, J = 2.2 Hz, 1H), 4.76 (s, 2H), 3.80 (s, 3H).
4.1.17.2 2,2,2-Trichloroethyl (3-(4-fluorophenoxy)-5-((2-methyl-
1-oxo-1,2,3,4-tetrahydroisoquinolin-6-yl)oxy)phenyl)carbamate
(27b).
1
Yield: 96%, H NMR (400 MHz, CDCl3) δ 8.04 (d, J = 8.5
Hz, 1H), 7.24 (s, 1H), 7.14 – 6.96 (m, 4H), 6.96 – 6.82 (m, 3H),
6.81 – 6.75 (m, 1H), 6.39 (s, 1H), 4.77 (s, 2H), 3.55 (t, J = 6.6
Hz, 2H), 3.14 (s, 3H), 2.96 (t, J = 6.5 Hz, 2H).
4.1.17.3 2,2,2-Trichloroethyl (3-(4-fluorophenoxy)-5-((1-oxo-
1,2,3,4-tetrahydroisoquinolin-6-yl)oxy)phenyl)carbamate (27c).
1
Yield: 95%, H NMR (400 MHz, CDCl3) δ 8.05 – 7.99 (m,
1H), 7.16 (s, 1H), 7.09 – 7.00 (m, 4H), 6.96 – 6.91 (m, 1H), 6.91
– 6.86 (m, 2H), 6.84 – 6.79 (m, 1H), 6.41 (s, 1H), 6.13 (s, 1H),
4.78 (s, 2H), 3.60 – 3.51 (m, 2H), 2.96 (t, J = 6.1 Hz, 2H).
4.1.18.3 4-(2-Ethylbutyl)-N-(3-(4-fluorophenoxy)-5-((1-oxo-
1,2,3,4-tetrahydroisoquinolin-6-yl)oxy)phenyl)-4-hydroxypiperi-
dine-1-carboxamide (28c).
Compound 28c was eluted with ethyl acetate as white solid.
Yield: 26%, M.P. 81 – 83 °C. 1H NMR (400 MHz, CDCl3) δ 7.99
(d, J = 8.5 Hz, 1H), 7.07 – 6.96 (m, 3H), 6.96 – 6.89 (m, 1H),
6.88 – 6.77 (m, 2H), 6.56 (s, 1H), 6.33 (s, 1H), 5.95 (s, 1H), 3.85
– 3.71 (m, 2H), 3.61 – 3.48 (m, 2H), 3.34 – 3.18 (m, 2H), 2.99 –
4.1.17.4 2,2,2-Trichloroethyl (3-(4-((2-fluoroethyl)carbamoyl)p-
henoxy)-5-(4-fluorophenoxy)phenyl)carbamate (27d).
1
Yield: 90%. H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 8.7
Hz, 2H), 7.10 – 6.97 (m, 7H), 6.88 (s, 1H), 6.83 (s, 1H), 6.45 (s,

2.87 (m, 2H), 1.65 – 1.52 (m, 5H), 1.44 – 1.31 (m, 7H), 1.15 (s,
1H), 0.84 (s, 6H). 13C NMR (101 MHz, CDCl3) δ 166.00, 160.41,
159.24, 158.97 (d, J = 243.4 Hz), 157.03, 154.45, 152.13 (d, J =
2.0 Hz), 142.15, 141.14, 130.07, 123.60, 120.88 (d, J = 8.1 Hz),
116.52, 116.32 (d, J = 23.2 Hz), 116.30, 105.61, 105.10, 103.90,
70.13, 46.72, 40.41, 40.09, 37.06, 35.32, 28.43, 27.27, 10.80.
HRMS (ESI) m/z [M + Na]+ calcd. for C33H38FN3NaO5
598.2688, found 598.2680.
2H), 3.40 – 3.18 (m, 2H), 3.04 (s, 3H), 1.95 – 1.75 (m, 1H), 1.70
– 1.50 (m, 4H), 1.46 – 1.28 (m, 3H), 0.99 (s, 6H). 13C NMR (101
MHz, CDCl3) δ 159.10 (d, J = 244.4 Hz), 159.07, 154.15, 151.93
(d, J = 3.0 Hz), 145.75, 141.99, 137.46, 132.85, 127.99, 127.83,
120.95 (d, J = 8.1 Hz), 118.96, 116.95, 116.48 (d, J = 23.2 Hz),
109.95, 70.13, 51.94, 44.53, 40.43, 37.10, 24.84, 23.27. HRMS
(ESI) m/z [M + Na]+ calcd. for C29H33FN2NaO5S2 595.1707,
found 595.1693.
4.1.18.4 4-(2-Ethylbutyl)-N-(3-(4-((2-fluoroethyl)carbamoyl)phe-
noxy)-5-(4-fluorophenoxy)phenyl)-4-hydroxypiperidine-1-
carboxamide (28d).
4.2 Sphingosine-1-phosphate receptors binding assay
[32P]S1P was freshly prepared by following our previously
published protocol26 and dissolved in DMSO, which was further
diluted to 0.3-0.6 nM with assay buffer (50 mM HEPES-Na, pH
7.5, 5 mM MgCl2, 1 mM CaCl2, 0.5% fatty acid-free BSA). The
test compounds were dissolved in DMSO and diluted into six
different concentrations (0.03, 0.3, 3.0, 30, 300, and 3000 nM)
with assay buffer. The commercial cell membranes expressing
recombinant human S1PRs were diluted with assay buffer to
make a 20-40 µg/mL of solution. To a 96-well plate was added
50 µL of cell membranes, 50 µL of test compounds, and 50 µL of
[32P]S1P. Each well has a final volume of 150 µL containing 0.1-
0.2 nM of [32P]S1P, 1-2 µg of membrane protein (S1PRs), and
different concentrations (0.01-1000 nM) of test compounds. The
plate was incubated for 60 min at room temperature with shaking
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 a total of five
washes. The filter bound radionuclide was measured by a
Beckman LS 3801 scintillation counter using Cherenkov
counting. The IC50 values were fitted from GraphPad Prism 6
using one site Nonlinear Regression.
Compound 28d was eluted with hexane/ethyl acetate (7/3,
V/V) as white solid. Yield: 88%, M.P. 79 – 82 °C. 1H NMR (400
MHz, CDCl3) δ 7.67 (d, J = 7.6 Hz, 2H), 7.14 – 6.93 (m, 6H),
6.90 (s, 1H), 6.86 – 6.76 (m, 3H), 6.31 (s, 1H), 4.68 – 4.46 (m,
2H), 3.84 – 3.62 (m, 4H), 3.26 – 3.12 (m, 2H), 1.58 – 1.44 (m,
4H), 1.38 – 1.22 (m, 8H), 0.88 – 0.78 (m, 6H). 13C NMR (101
MHz, DMSO) δ 166.16, 159.35, 158.84 (d, J = 241.39 Hz),
158.94, 157.41, 154.60, 152.46, 144.02, 129.81, 129.59, 121.50
(d, J = 8.1 Hz), 118.39, 117.00 (d, J = 24.2 Hz), 104.51, 104.03,
102.45, 82.60 (d, J = 166.7 Hz), 68.88, 46.52, 40.38, 37.18,
35.07, 27.18, 11.15. HRMS (ESI) m/z [M + Na]+ calcd. for
C33H39F2N3NaO5 618.2750, found 618.2727.
4.1.18.5 4-(2-Ethylbutyl)-N-(3-(4-fluorophenoxy)-5-((4-
(methylsulfonyl)phenyl)thio)phenyl)-4-hydroxypiperidine-1-
carboxamide (28e).
Compound 28e was eluted with hexane/ethyl acetate (2/3,
V/V) as white solid. Yield: 27%, M.P. 78 – 80 °C. 1H NMR (400
MHz, CDCl3) δ 7.76 (d, J = 8.3 Hz, 2H), 7.31 (d, J = 8.2 Hz,
2H), 7.21 (s, 1H), 7.14 (s, 1H), 7.05 – 6.93 (m, 4H), 6.72 (s, 1H),
6.59 (s, 1H), 3.85 – 3.70 (m, 2H), 3.24 – 3.15 (m, 2H), 3.02 (s,
3H), 1.65 – 1.53 (m, 4H), 1.44 – 1.28 (m, 7H), 1.14 (s, 1H), 0.84
(t, J = 6.7 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 159.06 (d, J =
243.4 Hz), 159.00, 154.25, 151.94 (d, J = 2.0 Hz), 145.85,
142.15, 137.33, 132.65, 127.90, 127.79, 120.92 (d, J = 9.1 Hz),
119.08, 116.86, 116.45 (d, J = 24.2 Hz), 110.06, 70.11, 46.72,
44.50, 40.39, 37.03, 35.31, 27.28, 10.82. HRMS (ESI) m/z [M +
Na]+ calcd. for C31H37FN2NaO5S2 623.2020, found 623.2018.
Abbreviations
S1P, sphingosine 1-phosphate; S1PR, sphingosine 1-
phosphate receptor; CNS, central nervous system; HTS, high
throughput screening; BBB, blood-brain barrier; PET, positron
emission tomography; DMF, N,N-dimethylformamide; DMA,
N,N-dimethylacetamide; DIPEA, N,N-diisopropylethylamine;
DMSO, dimethyl sulfoxide; THF, tetrahydrofuran; RT, room
temperature; TLC, thin-layer chromatography
4.1.18.6 N-(3-(4-((2-fluoroethyl)carbamoyl)phenoxy)-5-(4-
fluorophenoxy)phenyl)-4-hydroxy-4-isobutylpiperidine-1-
carboxamide (28f).
Acknowledgements
This study was supported by the National Multiple Sclerosis
Society [RG150705331]; National Institutes of Health
[NS103988, NS075527, and EB025815]
Compound 28f was eluted with hexane/ethyl acetate (1/2,
V/V) as white solid. Yield: 70%, M.P. 81 – 83 °C. 1H NMR (400
MHz, CDCl3) δ 7.67 (d, J = 8.2 Hz, 2H), 7.06 – 6.90 (m, 7H),
6.86 – 6.75 (m, 3H), 6.31 (s, 1H), 4.68 – 4.46 (m, 1H), 4.55 –
4.46 (m, 1H), 3.82 – 3.60 (m, 4H), 3.19 (t, J = 11.5 Hz, 2H), 1.87
– 1.74 (m, 1H), 1.60 – 1.46 (m, 4H), 1.39 – 1.28 (m, 3H), 0.93 (d,
J = 6.5 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 167.44, 159.72,
159.28, 158.99 (d, J = 243.4 Hz), 157.35, 154.55, 152.04 (d, J =
2.0 Hz), 142.05, 128.97, 128.89, 120.96 (d, J = 8.1 Hz), 118.10,
116.34 (d, J = 23.2 Hz), 105.01, 104.83, 103.55, 82.54 (d, J =
167.7 Hz), 70.03, 51.86, 40.54, 40.31, 37.06, 24.84, 23.22.
HRMS (ESI) m/z [M + Na]+ calcd. for C31H35F2N3NaO5
590.2437, found 590.2430.
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Graphical Abstract
Design, synthesis, and in vitro bioactivity evaluation of
Leave this area blank for
abstract info.
fluorine-containing analogues for
sphingosine-1-phosphate 2 receptor
Zonghua Luoa , Hui Liua , Robyn S. Kleinb,c,d , and Zhude Tua*
Department of Radiology, Washington University School of Medicine, St. Louis, MO 63110,
a
USA
b Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110,
USA
c Department of Neuroscience, Washington University School of Medicine, St. Louis, MO
63110, USA
d Department of Pathology & Immunology, Washington University School of Medicine, St.
Louis, MO 63110, USA