Z. Li et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
5
0
0
ethanol (5 mL) and toluene (15 mL). After nitrogen substitution, Pd
PPh (0.05 equiv) was added. The reaction mixture was stirred at
0 °C under nitrogen atmosphere for 12 h. The reaction mixture
3-(4-((4 -Methyl-[1,1 -biphenyl]-2-yl)methoxy)phenyl)propanoic-
1
(
8
3
)
4
2,2-d
2
acid (5). Yield 71%; white solid, m.p. 112–113 °C. H NMR
) d: 12.12 (s, 1H), 7.68–7.52 (m, 1H), 7.47–
(300 MHz, DMSO d
6
was cooled, and water (15 mL) was added. The mixture was diluted
with ethyl acetate (15 mL), and the insoluble material was filtered
off through Celite. The organic layer of the filtrate was washed with
brine, dried over anhydrous sodium sulfate, and concentrated in
vacuo. The residue was purified by silica gel column chromatogra-
phy using a mixture of petroleum ether/ethyl acetate (10:1, v/v) as
eluent to afford the desired product 6a–d as a solid. To a solution of
7.35 (m, 2H), 7.35–7.26 (m, 3H), 7.22 (d, J = 8.0 Hz, 2H), 7.09 (d, J
= 8.5 Hz, 2H), 6.78 (d, J = 8.5 Hz, 2H), 4.89 (s, 2H), 2.72 (s, 2H),
2.32 (s, 3H). C NMR (75 MHz, DMSO d ) d: 174.27, 156.95,
6
1
3
141.94, 137.49, 137.05, 134.34, 133.51, 130.31, 130.09, 129.64,
129.34, 129.27, 128.66, 127.81, 114.93, 68.08, 29.88, 21.14. ESI-
ꢁ
MS m/z: 347.1 [MꢁH] . Anal. calcd. For C23
20 2 3
H D O : C, 79.28; H,
6.94; Found: C, 79.24; H, 6.95.
0
6
a–d (1 equiv) in MeOH (10 mL) and THF (20 mL) was added por-
3-(4-((4-Fluoro-[1,1 -biphenyl]-2-yl)methoxy)phenyl)propanoic-
1
tionwise sodium borohydride (3 equiv) at 0 °C and the mixture was
stirred at 0 °C for 30 min. The reaction mixture was pouring into
ice water (10 mL), and extracted with ethyl acetate (3 ꢂ 15 mL),
the organic fractions were combined, washed with saturated brine
2,2-d
2
acid (6). Yield 67%; white solid, m.p. 101–103 °C. H NMR
) d: 12.09 (s, 1H), 7.64, 7.62 (dd, J = 8.5, 6.1
(300 MHz, DMSO d
6
Hz, 1H), 7.51–7.35 (m, 5H), 7.33–7.15 (m, 2H), 7.09 (d, J = 8.6 Hz,
2H), 6.78 (d, J = 8.6 Hz, 2H), 4.86 (s, 2H), 2.72 (s, 2H). 13C NMR
(
2 ꢂ 15 mL) prior to drying over anhydrous sodium sulfate. After
6
(75 MHz, DMSO d ) d: 174.51, 156.83, 144.45, 144.34, 139.29,
filtration and concentrate using a rotary evaporator, the residue
was used in next step without further purification. To a solution
of the obtained solid (1 equiv) in dichloromethane (20 mL) was
slowly added thionyl chloride (6 equiv) and a catalytic amount of
DMF at room temperature. After stirring at 40 °C for 4 h, the reac-
tion was concentrated under reduced pressure. The residue was
purified by silica gel column chromatography using a mixture of
petroleum ether/ethyl acetate (20:1, v/v) as eluent to afford the
desired product.
133.63, 132.71, 132.59, 130.75, 129.67, 129.31, 128.88, 128.33,
ꢁ
117.01, 116.73, 114.96, 67.45, 29.88. ESI-MS m/z: 351.1 [MꢁH] .
17 2 3
Anal. calcd. For C22H D FO : C, 74.98; H, 6.01; Found: C, 74.95;
H, 6.02.
0
3-(4-([1,1 -Biphenyl]-2-ylmethoxy)phenyl)propanoic-2,2-d
2
acid
1
(7). Yield 74%; white solid, m.p. 117–119 °C. H NMR (300 MHz,
DMSO d ) d: 12.19 (s, 1H), 7.59 (d, J = 5.4 Hz, 1H), 7.48–7.31 (m,
8H), 7.09 (d, J = 8.2 Hz, 2H), 6.77 (d, J = 8.2 Hz, 2H), 4.90 (s, 2H),
6
1
3
6
2.72 (s, 2H). C NMR (75 MHz, DMSO d ) d: 174.31, 156.94,
0
0
2
-(Chloromethyl)-4-fluoro-4 -methyl-1,1 -biphenyl (7a). Yield
141.99, 140.43, 134.35, 133.53, 130.33, 130.11, 129.64, 129.37,
1
4
7
5%; H NMR (300 MHz, DMSO d
6
) d: 7.33 (d, J = 8.1 Hz, 2H),
128.74, 128.02, 127.81, 114.93, 68.07, 29.90. ESI-MS m/z: 333.1
ꢁ
.27–7.08 (m, 5H), 4.65 (s, 2H), 2.33 (s, 3H).
[MꢁH] . Anal. calcd. For C22
18 2 3
H D O : C, 79.02; H, 6.63; Found: C,
0
0
2
-(Chloromethyl)-4 -methyl-1,1 -biphenyl (7b). Yield 52%; 1H
79.05; H, 6.64.
NMR (300 MHz, DMSO d
2
(
6
) d: 7.69–7.53 (m, 1H), 7.46–7.34 (m,
H), 7.37–7.28 (m, 3H), 7.22 (d, J = 8.0 Hz, 2H), 4.65 (s, 2H), 2.32
s, 3H).
-(Chloromethyl)-4-fluoro-1,1 -biphenyl (7c). Yield 58%; H NMR
300 MHz, DMSO d ) d: 7.66, 7.64 (dd, J = 8.6, 6.2 Hz, 1H), 7.52–
.36 (m, 5H), 7.34–7.16 (m, 2H), 4.65 (s, 2H).
4.2. Biological methods
0
1
2
4.2.1. FFA4 b-arrestin recruitment assay
CHO cells expressing hFFA4 were plated in sterile poly-
coated 384-well microplates in 25 l of DME/F12 with 0.1% char-
(
6
D-lysine
7
l
0
2
-(Chloromethyl)-1,1 -biphenyl (7d). Yield 49%; 1H NMR (300
MHz, DMSO d ) d: 7.58 (d, J = 5.6 Hz, 1H), 7.49–7.32 (m, 8H), 4.65
coal/dexstran treated FBS. The plates were then incubated over-
night at 37 °C. On the day of the experiment, the growth media
6
(
s, 2H).
was removed from the assay plates and 25
ll of HBSS containing
0
.1% BSA is added to each well. Test compounds with various con-
4.1.3. General synthetic procedure for target compounds 4–7
centrations were added into the cells. Plates were then incubated
To a solution of 3a (1 equiv) and intermediates 7a–d (1 equiv)
for 90 min at 37 °C. Detection reagent is prepared as described
in acetonitrile was added K
2
CO
3
(2 equiv) and a catalytic amount
by the manufacturer and after the 90 min incubation, 12 ll is
of KI at room temperature. The reaction mixture was heated to
reflux with stirring overnight. Then the reaction mixture was
cooled to room temperature followed by filtration and the filtrate
was concentrated under vacuum. The residue was purified by silica
gel column chromatography using a mixture of petroleum ether/
ethyl acetate (4:1, v/v) as eluent to afford a white solid. To a solu-
added to each well. The plates are then incubated for 60 min at
room temperature in the dark. The plates are then read in a Perkin
Elmer Envision reader measuring luminescence (0.1 s reading).
Luminescence values are normalized to% activation using wells
that contain DMSO alone (0% activity) and wells that contained a
concentration of agonist known to maximally activate hFFA4
(100% activity). EC50 value of test compound was calculated by
GraphPad InStat version 5.00 (GraphPad software, San Diego, CA,
USA).
tion of the obtained solid (1 equiv) in 2:3:1 THF/MeOH/H
2
O (18
mL) was added LiOHꢀH O (3 equiv). After stirring at room temper-
2
ature for 4 h, the volatiles were removed under reduced pressure.
The residue was acidified with 1N hydrochloric acid solution, and
then filtered and the filter cake was washed with 5 mL of water,
dried in vacuum to afford a white powder. The white powder
was purified by column chromatography using a mixture of petro-
leum ether/ethyl acetate (2:1–1:2, v/v) as eluent to afford the tar-
get compounds as white solid.
2
+
4.2.2. Ca influx activity (FLIPR assay)
CHO cells expressing hFFA1 were seeded on 96-well tissue-cul-
ture plates (15 K cells per well) and incubated at 37 °C for 16 h
under 5% CO
was removed and washed with Hank’s Balanced Salt Solution
(100 L per well). Then, cells were incubated in buffer containing
2.5 g/mL Fluo 4-AM (fluorescent calcium indicator), 0.1% fatty
acid-free BSA and 2.5 mmol/L probenecid for 1 h at 37 °C. Test
compounds or -linolenic acid (Sigma) with various concentrations
2
prior to the assay. The medium in culture wells
0
0
3
-(4-((4-Fluoro-4 -methyl-[1,1 -biphenyl]-2-yl)methoxy)phenyl)
propanoic-2,2-d acid (4). Yield 78%; white solid, m.p. 105–106 °C.
H NMR (300 MHz, DMSO d ) d: 12.13 (s, 1H), 7.32 (d, J = 8.0 Hz,
H), 7.29–7.05 (m, 7H), 6.79 (d, J = 8.5 Hz, 2H), 4.85 (s, 2H), 2.71
s, 2H), 2.32 (s, 3H). 13C NMR (75 MHz, DMSO d
) d: 174.27,
l
2
l
1
6
2
(
c
2+
6
were added into the cells, and the Ca flux signals after addition
were recorded by the FLIPR Tetra system (Molecular Devices).
The hFFA1 agonistic activities of test compounds were expressed
1
1
2
56.85, 144.34, 139.59, 136.07, 134.54, 132.47, 130.76, 130.13,
29.67, 129.03, 128.65, 127.83, 115.67, 114.95, 68.08, 29.86,
ꢁ
2+
1.15. ESI-MS m/z: 365.1 [MꢁH] . Anal. calcd. For C23
H
19
D
2
3
FO :
as [(A ꢁ B)/(C ꢁ B)] ꢂ 100 (the increase of Ca flux signals (A) in
C, 75.39; H, 6.33; Found: C, 75.34; H, 6.32.
test compounds and (B) in vehicle, and (C) in 10 lM c-linolenic